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Effects of growth hormone-releasing hormone gene targeted ablation on ghrelin-induced feeding
Growth Hormone & IGF Research 37 (2017) 40–46
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Growth Hormone & IGF Research journal homepage: www.elsevier.com/locate/ghir
Effects of growth hormone-releasing hormone gene targeted ablation on ghrelin-induced feeding
MARK
Lucia Recinellaa, Sheila Leonea, Claudio Ferrantea, Annalisa Chiavarolia, Rugia Shohreha, Chiara Di Nisioa, Michele Vaccaa, Giustino Orlandoa,⁎, Roberto Salvatorib, Luigi Brunettia a b
Department of Pharmacy, G. d'Annunzio University, Chieti, Italy Division of Endocrinology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Growth hormone-releasing hormone knockout Ghrelin Feeding NPY AgRP CRH
Impairment of growth hormone (GH) signaling has been associated with increased feeding and adiposity. The gastric hormone ghrelin, in addition to its GH-secretagogue effects, stimulates food intake after both central and peripheral administration. In the present study we further investigated the feeding regulatory role of the ghrelinGH axis in a mouse model of isolated GH deficiency due to targeted ablation of the GH-releasing hormone (GHRH) gene [GHRH knockout (GHRHKO)]. We evaluated the effects of intracerebroventricular ghrelin administration on feeding behavior, related hypothalamic neuropeptides and neurotransmitters, and serum ghrelin levels in mice homozygous for GHRHKO allele (−/−) and heterozygous (+/−) control animals. Vehicletreated GHRHKO mice showed increased food intake compared to heterozygotes, associated with increased circulating ghrelin levels. Moreover, −/− mice showed elevated hypothalamic levels of neuropeptide Y (NPY), agouti-related peptide (AgRP) mRNAs and norepinephrine (NE) and decreased corticotropin-releasing hormone (CRH) mRNA levels. Ghrelin treatment significantly augmented food intake in both genotypes, but the relative increase compared to vehicle-treated animals was higher in −/− than +/− mice. In the hypothalamus, ghrelin increased AgRP and decreased CRH gene expression only in heterozygous mice, while it induced a significant reduction in proopiomelanocortin (POMC) mRNA levels in −/− mice. Ghrelin treatment also decreased hypothalamic serotonin (5-hydroxytriptamine, 5-HT) and dopamine (DA) levels in both genotypes. Additionally, we observed increased DA metabolism induced by ghrelin in both genotypes. In conclusion, dysregulation of the ghrelin-GHRH-GH axis in GHRHKO mice could lead to increased feeding secondary to elevated circulating levels of ghrelin, and the obesogenic phenotype is likely mediated by elevated NPY and AgRP, and decreased CRH gene expression in the hypothalamus.
1. Introduction Besides the well-known somatic growth stimulatory effects, growth hormone (GH) also plays a key role in energy balance [1]. In adipose tissue, GH stimulates lipolysis [2]. Accordingly, GH deficiency (GHD) is characterized by increased fat depots in both humans and mice [3,4]. The effects of GH on feeding have been investigated with inconsistent results [5–9]. We have previously reported that a mouse model of isolated GHD (IGHD) due to targeted ablation of the GH-releasing hormone gene (GHRH knockout, GHRHKO) shows increased food intake, visceral and subcutaneous fat depots and serum adiponectin levels [10]. Similarly, humans with IGHD due to a null mutation in the GHRH receptor gene have higher energy intake (when corrected by body weight) compared to controls [11]. The isolation of the gastric hormone ghrelin as the endogenous ⁎
ligand of the GH secretagogue receptor (GHSR) [12] has provided an intriguing dimension to the role of GH on feeding. GHSR's are expressed in the arcuate nucleus of the hypothalamus (ARC), as well as in other hypothalamic nuclei [13], and GHRH neurons are a major target of ghrelin within the ARC [14,15]. In addition to stimulating GHRH-GH axis, ghrelin has been shown to stimulate feeding and body weight gain when administered either centrally or peripherally [16–18]. The ARC is a major site for transducing peripheral afferents into the neural network involved in feeding control [19]. The ARC includes two populations of peptidergic neurons expressing either neuropeptide Y (NPY)/agouti-related peptide (AgRP) that stimulate food intake, or proopiomelanocortin (POMC)/cocaine- and amphetamine-regulated transcript (CART), which have anorexigenic effects. Both neuronal populations project to neurons located in the lateral hypothalamus (LH) and paraventricular nucleus (PVN), which express orexin and
Corresponding author at: Department of Pharmacy, G. d'Annunzio University, via dei Vestini 31, 66013 Chieti, Italy. E-mail address: [email protected] (G. Orlando).
http://dx.doi.org/10.1016/j.ghir.2017.10.007 Received 1 August 2017; Received in revised form 23 October 2017; Accepted 25 October 2017 1096-6374/ © 2017 Elsevier Ltd. All rights reserved.
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infection. Immediately after surgery, the mice were injected subcutaneously with 0.5 ml of sterile saline solution and 0.5 ml of 5% glucose solution, and intraperitoneally with amoxicillin/clavulanic acid (30 mg/kg/day). Beginning one week after surgery, at 09:00 a.m., 2.5 month aged −/− (n = 12) and +/− (n = 12) mice were divided into two groups and injected into third ventricle with either ghrelin (3 nmol, 1 μl) or vehicle (saline, 1 μl). Food intake was recorded 1, 2, 8 and 24 h after treatment in each group, by subtracting from supplied pellet the residual in the dispenser and sawdust bedding. Because of the significant difference in total body weight (TBW) between −/− and +/− mice, food intake in grams of chow was normalized to TBW (% of TBW). Finally, animals were euthanized by CO2 inhalation (100% CO2 at a flow rate of 20% of the chamber volume per minute) and blood was collected by cardiac puncture.
corticotropin-releasing hormone (CRH), and drive orexigenic or anorexigenic behavior, respectively [20]. Ghrelin expressing neurons target these hypothalamic peptidergic circuits [21,22], adding to systemicderived ghrelin in modulating feeding control. GHD in GHRHKO animals could possibly involve changes in ghrelin secretion, with consequent effects on feeding control. Hypothalamic aminergic neurotransmitters also play a role in food intake regulation and we have previously reported that adipokines and gut-derived hormones which are known to affect feeding, also modulate hypothalamic dopamine (DA), norepinephrine (NE) and serotonin (5hydroxitriptamine, 5-HT) levels [23–26]. The aim of the present study was to further investigate the roles played by ghrelin in GHRH deficiency-associated dysregulation of feeding. We evaluated food intake, serum ghrelin levels and mRNA levels of CART, CRH, POMC, AgRP, NPY and orexin-A in the hypothalamus, after either vehicle or exogenous ghrelin intracerebroventricular (i.c.v.) administration. We also investigated the effects of exogenous ghrelin on hypothalamic neurotransmitters DA, NE, 5-HT, and their metabolites homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5HIIA) concentrations.
2.3. Serum ghrelin levels Serum total ghrelin levels were measured using the human/mouse/ rat ghrelin enzyme immunoassay (EIA) kit (#K4790-100, Enzo Life Sciences AG, Lausen, Switzerland), following the manufacturer's instruction manual. The sensitivity of the EIA kit was 161 pg/ml. Samples were analyzed in duplicate and the ghrelin levels were determined according to the calibration curves established from standard. The intra and inter-assay coefficients of variation were < 10% and < 15%, respectively.
2. Materials and methods 2.1. Animals and drugs Homozygous mice carrying a targeted ablation of the GHRH gene were previously described [27]. We employed adult (2.5 months old) (10–13 g) homozygous GHRHKO (−/−) male mice (n = 12) and adult (2.5 months old) (20–24 g) male mice heterozygous for the GHRHKO allele (+/−) (n = 12) as control group. GHRHKO offsprings were generated by mating heterozygous males and females, as previously reported [27]. Heterozygous mice were used as control because no significant difference in body weight was observed between +/+ and +/− animals from the 2nd to the 12th week of life [27] despite a modest difference in circulating insulin-like growth factor 1 (IGF1) levels [27]. Indeed, we did not observe any behavioral and neurotransmitter level difference in multiple brain areas between homozygous normal and heterozygous animals (supplementary data). The animals were housed in Plexiglas cages (35 × 20 × 13 cm), and maintained in acclimatized colony rooms (21 ± 1 °C; 55 ± 5% humidity), on a 14 h/10 h light/dark cycle, with continuous ad libitum access to water and food. Mice were fed with a standard rodent chow (Prolab RMH2500, PMI Nutrition International, Brentwood, MO). Housing conditions and experimentation procedures were strictly in agreement with the European Community ethical regulations (EU Directive n. 63/2010) on the care of animals for scientific research, and approved by Italian Health Ministry (Project n. 955/2016-PR). Rat recombinant ghrelin (1 mg/vial) was purchased from Vincibiochem [Vinci (FI), Italy] and diluted in saline.
2.4. RNA extraction, reverse transcription and real-time reverse transcription polymerase chain reaction (real-time RT PCR) Immediately after sacrifice, brains were rapidly removed and individual hypothalami immediately dissected and stored in RNAlater solution (Life Technologies, Carlsbad, CA, USA) at − 20 °C until further processing. Total RNA was extracted from the hypothalami using TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA), as previously reported [28]. One microgram of total RNA extracted from each sample in a 20 μl reaction volume was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's manual. Reactions were incubated in a 2720 Thermal Cycler (Life Technologies, Carlsbad, CA, USA) initially at 25 °C for 10 min, then at 37 °C for 120 min and finally at 85 °C for 5 s. Gene expression was determined by quantitative real-time PCR using TaqMan probe-based chemistry (Life Technologies, Carlsbad, CA, USA). Reactions were performed in MicroAmp Fast Optic 96-well Reaction Plates (Life Technologies, Carlsbad, CA, USA) on an ABI PRISM 7900 HT Fast Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). PCR primers and TaqMan probes were obtained from Life Technologies (Assays-on-Demand Gene Expression Products, Mm00475829_g1 for agouti-related peptide gene, Mm03048253_m1 for neuropeptide Y gene, Mm01964031_s1 for orexin-A gene, Mm00489086_m1 for cocaine- and amphetamine-regulated transcript gene, Mm01293920_s1 for corticotrophin releasing hormone gene, Mm00435874_m1 for proopiomelanocortin gene and Mm00607939_s1 for β-actin gene). β-Actin was used as the housekeeping gene. Data were elaborated with the Sequence Detection System (SDS) software version 2.3 (Life Technologies, Carlsbad, CA, USA). The comparative 2− ΔΔCt method was used to quantify the relative abundance of mRNA and then determine the relative changes in individual gene expression (relative quantification) [29].
2.2. In vivo intracerebroventricular treatment Mice were anesthetized by intraperitoneal injection of ketamine/ xylazine (50 and 5 mg/kg, respectively), and placed in a stereotaxic instrument (David Kopf Instruments, Tujunga, CA). Coordinates for placement of a cannula into the third ventricle were as follows: anteroposterior, + 0.3 mm anterior to the bregma; mediolateral, + 1 mm, right to the bregma; dorsoventral, 3 mm. The site for the cannula was marked. Small holes were drilled into the skull and stainless-steel screws were placed. A hole was drilled through the skull, in the marked site, for the placement of the 24-gauge stainless-steel cannula (1.2 cm long), sterilized in autoclave 2 h before surgery. Once in place, the cannula was attached to the skull using dental cement (Formatray, Salerno, Italy). Sterile obturators were inserted into the cannulas to prevent them from clogging and to reduce the potential for brain
2.5. Hypothalamic neurotransmitter extraction and high performance liquid chromatography (HPLC) determination Immediately after sacrifice, individual hypothalami were rapidly removed and subjected to biogenic amine and metabolite extractive 41
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Fig. 1. Effects of acute i.c.v. ghrelin injection on food intake in ad libitum-fed −/− mice and control +/− animals. Vehicle or ghrelin (3 nmol) was administered by i.c.v. injections, during the light phase, at 09:00 a.m. Food consumption was recorded 24 h after treatment in each group of mice and normalized to total body weight (TBW). Values are given as means ± S.E.M. for each experimental group (n = 6). Vehicle-treated −/− mice consumed more food per TBW than vehicle-treated +/− animals (ANOVA, P < 0.0001; post hoc **P < 0.01 vs. +/− vehicle). Compared to vehicle, ghrelin treatment significantly increased food intake in both genotypes (ANOVA, P < 0.0001; post hoc ***P < 0.001 vs. vehicle-treated gene-matched controls), but the increased feeding response to ghrelin with respect to vehicle-treated animals was higher in −/− than +/− mice (ANOVA, P < 0.0001; post hoc #P < 0.001 vs. +/− ghrelin).
analysis, 1.00 (calibrator sample) was obviously considered the theoretical mean for the comparison. Gaussian distribution of data was assessed by D'Agostino and Pearson omnibus normality test. Differences were considered to be significant when P was < 0.05. As previously reported [30], we calculated the animals randomized for each experimental group on the basis of the “Resource Equation” N = (E + T) / T (10 ≤ E ≤ 20), where E, N and T represent the numbers of degrees of freedom in an ANOVA, animals and treatment, respectively. In our experiments E was 20, for behavioral evaluation. On the other hand, E was 10, for biochemical evaluations. Any sample size, which keeps E between 10 and 20, should be considered as an adequate. If E is < 10 then adding more animals will increase the chance of getting more significant result, but if it is > 20 then adding more animals will not increase the chance of getting significant results [31]. Considering that T value was 4, as regards to behavioral and ghrelin level evaluations, N value was 6, in both genotypes. In agreement with the recognized principles of “Replacement, Reduction and Refinement of Animals in Research”, N value was 3 for neurotransmitter and neuropeptide activity evaluations, requiring different experimental paradigms of extraction and quantification, without adding more animals to the experimental procedures.
procedures [25]. Briefly, samples were treated with 1 ml of 0.05 N perchloric acid containing 0.004% sodium EDTA and 0.010% sodium bisulfite and centrifuged at 4500g for 10 min. The supernatant was filtered on 0.45 μm PTFE sterile filters (Whatman) and directly injected for HPLC. Analyte recovery was satisfactory (≥90%) and reproducible, with percentage relative standard deviation ≤ 10%. The HPLC apparatus consisting of a Jasco (Tokyo, Japan) PU-2080 chromatographic pump and an ESA (Chelmsford, MA, USA) Coulochem III coulometric detector, equipped with microdialysis cell (ESA-5014b) porous graphite working electrode and solid state palladium reference electrode. The analytical cell was set at − 0.150 V, for detector 1 and at +0.300 V, for detector 2, with a range of 100 nA. The chromatograms were monitored at the analytical detector 2. Integration was performed by Jasco Borwin Chromatography software, version 1.5. The chromatographic separation was performed by isocratic elution on Phenomenex Kinetex reverse phase column (C18, 150 mm × 4.6 mm i.d., 2.6 μm). The mobile phase was (10:90, v/v) acetonitrile and 75 mM pH 3.00 phosphate buffer containing octanesulfonic acid 1.8 mM, EDTA 30 μM and triethylamine 0.015% (v/v). Flow rate was 0.6 ml/min and the samples were manually injected through a 20 μl loop. Neurotransmitter and metabolite peaks were identified by comparison with the retention time of pure standard. Neurotransmitter and metabolite concentrations in the samples were calculated by linear regression curve (y = bx + m) obtained with standard. Neither internal nor external standard were necessary for analyte quantification, in the hypothalamus homogenate, and all tests performed for method validation yielded results in accordance to limits indicated in official guidelines for applicability in laboratory trials. The standard stock solutions of DA, NE, 5-HT, HVA, DOPAC and 5-HIIA at 2 mg/ml were prepared in bidistilled water containing 0.004% EDTA and 0.010% sodium bisulfite. The stock solutions were stored at 4 °C. Work solutions (1.25–20.00 ng/ml) were obtained daily progressively diluting stock solutions in mobile phase.
3. Results 3.1. Food intake and serum ghrelin levels As previously reported [10], when food intake was normalized as percentage of TBW, vehicle-treated −/− mice consumed more food than +/− animals (ANOVA, P < 0.0001; post hoc **P < 0.01 vs. +/− vehicle). Ghrelin treatment significantly augmented food intake both in −/− and +/− mice 24 h following injection (ANOVA, P < 0.0001; post hoc ***P < 0.001 vs. vehicle-treated gene-matched controls), and the relative increase compared to vehicle-treated animals was higher in −/− than +/− mice (ANOVA, P < 0.0001; post hoc #P < 0.001 vs. +/− ghrelin) (Fig. 1). Conversely, no change in feeding was induced by ghrelin 1, 2 and 8 h post injection [data not shown]. As compared to +/− animals, serum ghrelin levels were higher in vehicle-treated −/− mice. In addition, ghrelin administration resulted in increased serum levels of ghrelin in both genotypes [(+/− vehicle 4.45 ± 0.15 ng/ml; −/− vehicle 6.15 ± 0.28**ng/ ml; +/− ghrelin 20.75 ± 1.23***ng/ml; −/− ghrelin
2.6. Statistical analysis Statistical analysis was performed using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, CA, USA). All data were collected from each of the 24 animals used in the experimental procedure and means ± S.E.M. were collected for each experimental group and analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. As for gene expression 42
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Fig. 2. Relative gene expression of hypothalamic agouti-related peptide (AgRP) (A), neuropeptide Y (NPY) (B), orexin-A (C), cocaine- and amphetamine-regulated transcript (CART) peptide (D), corticotropin-releasing hormone (CRH) (E) and proopiomelanocortin (POMC) (F), 24 h after treatment with either vehicle or ghrelin in −/− mice and control +/− animals, as determined by real-time RT PCR. Data were calculated using the 2− ΔΔCt method; they were normalized to β-actin mRNA levels and then expressed as relative to vehicle-treated +/− mice (calibrator sample, defined as 1.00). Values are given as means ± S.E.M. for each experimental group. Vehicle-treated −/− mice had higher AgRP (ANOVA, P < 0.0005; post hoc ***P < 0.001 vs. +/− vehicle) (A) and NPY (ANOVA, P < 0.0001; post hoc ***P < 0.001 vs. +/− vehicle) (B) and lower CRH (ANOVA, P < 0.05; post hoc *P < 0.05 vs. +/− vehicle) (F) gene expression than +/− animals. Ghrelin treatment decreased POMC mRNA levels (ANOVA, P < 0.001; post hoc ***P < 0.001 vs. −/− vehicle) (E) in −/− mice with respect to vehicle. On the other hand, ghrelin increased AgRP (ANOVA, P < 0.001; post hoc *P < 0.05 vs. +/− vehicle) (A) and decreased CRH (ANOVA, P < 0.05; post hoc *P < 0.05 vs. +/− vehicle) (F) gene expression in +/− mice.
23.68 ± 1.45***ng/ml) ANOVA, P < 0.0001; post hoc **P < 0.01 vs. +/− vehicle, ***P < 0.001 vs. vehicle-treated gene-matched controls].
***P < 0.001 vs. +/− vehicle) (Fig. 2A) and NPY (ANOVA, P < 0.0001; post hoc ***P < 0.001 vs. +/− vehicle) (Fig. 2B), and decreased CRH (ANOVA, P < 0.05; post hoc *P < 0.05 vs. +/− vehicle) (Fig. 2F) gene expression levels. No genotype-related differences were observed in orexin-A, CART and POMC mRNA levels (Fig. 2C–E). Ghrelin treatment, while stimulating food intake in both genotypes, differently affected hypothalamic peptide gene expression. In −/− mice, ghrelin induced a significant reduction in POMC mRNA levels
3.2. Hypothalamic peptide gene expression As compared to vehicle-treated +/− animals, vehicle-treated −/− mice had increased AgRP (ANOVA, P < 0.0005; post hoc 43
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A
C
B
Fig. 3. Effects of acute i.c.v. ghrelin injection in ad libitum-fed −/− mice and control +/− animals on hypothalamic monoamines (ng/mg wet tissue). Dopamine (DA) (A), norepinephrine (NE) (B) and serotonin (5-hydrohytryptamine, 5-HT) (C) levels are expressed as mean ± S.E.M. for each experimental group. Compared to vehicle-treated +/− animals, −/− vehicle-treated mice showed higher NE levels (ANOVA, P < 0.05; post hoc *P < 0.05 vs. +/− vehicle) than their respective +/− littermates (B). Compared to vehicle, ghrelin treatment decreased DA (ANOVA, P < 0.001; post hoc **P < 0.01 vs. vehicle-treated gene-matched controls) (A) and 5-HT levels (ANOVA, P < 0.01; post hoc *P < 0.05 vs. vehicletreated gene-matched controls) (C) in both genotypes leaving unaffected NE levels (ANOVA, P < 0.05; post hoc #P < 0.05 vs. +/− ghrelin) (B).
detected on 5-HIIA/5-HT ratio values (+/− vehicle 1.05 ± 0.11; −/− vehicle 1.043 ± 0.02; +/− ghrelin 1.01 ± 0.08; −/− ghrelin 0.97 ± 0.20).
compared to vehicle (ANOVA, P < 0.001; post hoc ***P < 0.001 vs. −/− vehicle) (Fig. 2E), while it did not affect the other neuropeptides. On the other hand, in +/− mice, ghrelin administration increased AgRP (ANOVA, P < 0.001; post hoc *P < 0.05 vs. +/− vehicle) (Fig. 2A) and decreased CRH (ANOVA, P < 0.05; post hoc *P < 0.05 vs. +/− vehicle) (Fig. 2F) gene expression, leaving unaffected the other neuropeptides.
4. Discussion In confirming our previous findings showing that GHRHKO mice have increased food intake compared to +/− animals (Fig. 1), the present work demonstrates that GHRH homozygous gene deletion leads to increased circulating ghrelin levels and a characteristic pattern of neuropeptide and neurotransmitter signaling in the hypothalamus. The control of pituitary GH secretion by ghrelin is finely tuned by negative feedback regulatory loops. Decreased circulating ghrelin levels have been shown in acromegalic patients compared to healthy subjects [32,33], and after administration of exogenous GH, while plasma ghrelin is increased by hypophysectomy, in rats [34]. Accordingly, we hypothesized that in GHRHKO mice ghrelin levels could be upregulated due to decreased inhibitory feedback by GH and IGF1, and that this could play a role in increasing feeding and adipose tissue stores, possibly through modulation of neuropeptides and neurotransmitters in the hypothalamus. Our results show that GHRHKO mice have higher serum ghrelin levels, underlying their increased food intake as compared to heterozygous mice. Exogenous ghrelin administration further increase feeding in both genotypes, with a higher incremental effect in GHRHKO animals (Fig. 1). Our data also show that the increased feeding phenotype by GHRHKO mice could be related to specific involvement of neural pathways in the hypothalamus, which are also affected by
3.3. Hypothalamic aminergic neurotransmitters and metabolites Compared to vehicle-treated heterozygous controls, −/− vehicletreated mice had higher NE concentrations, in the hypothalamus (ANOVA, P < 0.05; post hoc *P < 0.05 vs. +/− vehicle) (Fig. 3B) while no genotype-related differences were observed in DA and 5-HT levels. Ghrelin treatment decreased DA (ANOVA, P < 0.001; post hoc **P < 0.01 vs. vehicle-treated gene-matched controls) (Fig. 3A) and 5HT levels (ANOVA, P < 0.01; post hoc *P < 0.05 vs. vehicle-treated gene-matched controls) (Fig. 3C) in both genotypes, leaving unaffected NE levels. Additionally, we did not observe any difference between vehicletreated +/− and vehicle-treated −/− mice in DA and 5-HT metabolism, evaluated as HVA/DOPAC and 5-HIIA/5-HT ratio values, respectively. Compared to vehicle administration, ghrelin increased HVA/ DOPAC ratio values, in both genotypes [(+/− vehicle 0.74 ± 0.03; −/− vehicle 0.61 ± 0.05; +/− ghrelin 2.38 ± 0.06**; −/− ghrelin 2.12 ± 0.10**) ANOVA, P < 0.0001; post hoc **P < 0.01 vs. vehicle-treated gene-matched controls]. Finally, no effects were 44
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pathways, in the hypothalamus [49]. The existence of a negative feedback system linking brain serotonin levels to plasma ghrelin in the regulation of energy homeostasis in mice has been suggested [44]. Pretreatment with 5-HT injected into the PVN was found to inhibit feeding stimulated by ghrelin injected into the PVN [45]. We did not find any significant genotype-related difference in vehicle-treated mice in hypothalamic 5-HT levels and 5-HIIA/5-HT ratio. Similarly, ghrelin injection reduced hypothalamic 5-HT levels (Fig. 3C), without any effect on 5-HT metabolism, evaluated as 5-HIIA/ 5-HT ratio, to a similar degree in both genotypes. Considering the inhibitory role of 5-HT on feeding, our findings showing decreased 5-HT levels after ghrelin treatment could be related to the orexigenic effects of ghrelin in both genotypes. In conclusion, dysregulation of the ghrelin-GHRH-GH axis in GHRHKO mice leads to increased feeding, likely caused by increased circulating ghrelin levels. The obesogenic phenotype could be mediated by elevated NPY and AgRP, and decreased CRH gene expression, together with increased NE levels in the hypothalamus.
exogenous ghrelin administration. Our experiments show that vehicle-treated GHRH −/− mice have a significant higher increase in NPY and AgRP (Fig. 2A–B), and decrease in CRH (Fig. 2F) mRNAs levels in the hypothalamus when compared to +/− animals. Considering that NPY and AgRP play a feeding stimulatory role, while CRH reduces feeding, their reciprocal modulation in −/− animals could be related to their increased endogenous ghrelin levels as compared to +/− controls. Ghrelin has been shown to stimulate feeding in wild type animals, possibly through increased NPY and AgRP levels [16,17,35,37,38]; moreover, feeding stimulation by ghrelin is lost in AgRP and NPY double KO mice [36]. Additionally, electrophysiological approaches have shown that ghrelin can activate NPY and AgRP co-expressing neurons and simultaneously reduce the activity of POMC neurons [21]. Our findings further show that exogenous ghrelin administration stimulates AgRP and decreases CRH gene expression but only in heterozygous and not in −/− mice. The latter genotype is characterized by basally increased AgRP and decreased CRH mRNA levels (as seen in vehicle-treated animals), possibly due to increased endogenous ghrelin concentrations, and this may explain why these transmitters cannot be further modified by exogenous ghrelin administration. On the other hand, the higher feeding response to ghrelin shown by −/− animals could be related to the decrease in POMC gene expression that we have observed being induced by ghrelin in −/− animals but not in heterozygotes. The role of POMC-derived peptides is somewhat controversial, since both the orexigenic β-endorphin and the anorexigenic α-MSH could be processed from POMC gene transcript [19]. Previous studies have hypothesized that increased feeding by ghrelin could be also mediated by decreased melanocortin signaling through central upregulation of α-MSH degrading enzyme prolyl carboxypeptidase [39]. Furthermore, NPY and AgRP co-expressing neurons stimulated by ghrelin could exert unidirectional inhibitory tone on POMC neurons [40,41]. We also found increased NE levels in vehicle-treated −/− mice compared to +/− controls (Fig. 3B). The role of NE as a central transducer of appetite/satiety signals is controversial, with either inhibiting or stimulating effects, possibly related to the activation of α1or α2-adrenoceptors, respectively [42]. The anorectic effects of central and peripheral leptin administration have been linked to a significant reduction in hypothalamic NE concentration in several hypothalamic nuclei, such as ARC, PVN and dorsomedial nucleus in the rat [43]. Moreover, elevated circulating levels of NE were found in ob/ob leptindeficient mice, suggesting an inverse correlation between NE and leptin as one of the mechanisms of feeding regulation by this peptide [20]. We have previously found that omentin-1, a fat depot-specific secretory protein stimulating feeding in the rat, also increases NE synthesis and release, in the hypothalamus [25]. Consequently, increased feeding behavior observed in vehicle-treated −/− mice could also be driven by augmented NE levels in the hypothalamus. However, ghrelin injection did not modify hypothalamic NE levels, compared to vehicle-treated gene-matched controls. The role of DA in feeding control is still unsettled, with studies showing both inhibitory and stimulatory effects. On one side, DA stimulates feeding after its injection into the lateral hypothalamus [46]. On the other hand, DA administration into the perifornical hypothalamus produces anorexigenic effects [47], and increased DA transmission is associated with the anorectic effects of amphetamines [48]. Our findings show no difference on hypothalamic DA levels in vehicletreated −/− mice compared to +/− controls. In addition, ghrelin administration decreases DA in both strains of mice. We also found that ghrelin treatment is associated to increased HVA/DOPAC ratio, which could be related to increased DA metabolism induced by the peptide. On the other hand, we did not observe any direct influence by the peptide on hypothalamic DA release, in vitro [23]. Nevertheless, our findings of increased AgRP and reduced POMC gene expression in +/− and −/− mice, respectively, following ghrelin injection further support a possible interaction between melanocortin and dopaminergic
Conflict of interest statement The authors declare no conflict interest. Funding This work was supported by grants from the Italian Ministry of University (FAR 2016). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ghir.2017.10.007. References [1] D. Le Roith, C. Bondy, S. Yakar, J.L. Liu, A. Butler, The somatomedin hypothesis: 2001, Endocr. Rev. 22 (2001) 53–74. [2] H.M. Goodman, Y. Schwartz, L.R. Tai, E. Gorin, Actions of growth hormone on adipose tissue: possible involvement of autocrine or paracrine factors, Acta Paediatr. Scand. Suppl. 367 (1990) 132–136. [3] M. Alba, D. Fintini, R. Salvatori, Effects of recombinant mouse growth hormone treatment on growth and body composition in GHRH knockout mice, Growth Hormon. IGF Res. 15 (2005) 275–282. [4] E. Gomes-Santos, R. Salvatori, T.O. Ferrão, C.R. Oliveira, R.D. Diniz, J.A. Santana, F.A. Pereira, R.A. Barbosa, A.H. Souza, E.V. Melo, C.C. Epitácio-Pereira, A.A. Oliveira-Santos, I.A. Oliveira, J.A. Machado, F.J. Santana-Júnior, J.A. BarretoFilho, M.H. Aguiar-Oliveira, Increased visceral adiposity and cortisol to cortisone ratio in adults with congenital lifetime isolated GH deficiency, J. Clin. Endocrinol. Metab. 99 (2014) 3285–3289. [5] M.J. Azain, T.J. Roberts, R.J. Martin, T.R. Kasser, Comparison of daily versus continuous administration of somatotropin on growth rate, feed intake, and body composition in intact female rats, J. Anim. Sci. 73 (1995) 1019–1029. [6] M. Bohlooly-Y, B. Olsson, C.E. Bruder, D. Lindén, K. Sjögren, M. Bjursell, E. Egecioglu, L. Svensson, P. Brodin, J.C. Waterton, O.G. Isaksson, F. Sundler, B. Ahrén, C. Ohlsson, J. Oscarsson, J. Törnell, Growth hormone overexpression in the central nervous system results in hyperphagia-induced obesity associated with insulin resistance and dyslipidemia, Diabetes 54 (2005) 51–62. [7] J. Klindt, J.T. Yen, F.C. Buonomo, A.J. Roberts, T. Wise, Growth, body composition, and endocrine responses to chronic administration of insulin-like growth factor I and(or) porcine growth hormone in pigs, J. Anim. Sci. 76 (1998) 2368–2381. [8] K. Malmlöf, N. Din, T. Johansen, S.B. Pedersen, Growth hormone affects both adiposity and voluntary food intake in old and obese female rats, Eur. J. Endocrinol. 146 (2002) 121–128. [9] T.J. Roberts, M.J. Azain, Somatotropin treatment reduces energy intake without altering protein intake in pigs selecting between high and low protein diets, J. Nutr. 127 (1997) 2047–2053. [10] L. Recinella, R. Shohreh, R. Salvatori, G. Orlando, M. Vacca, L. Brunetti, Effects of isolated GH deficiency on adipose tissue, feeding and adipokines in mice, Growth Hormon. IGF Res. 23 (2013) 237–242. [11] A.A. Oliveira-Santos, R. Salvatori, E. Gomes-Santos, J.A. Santana, Â.C. Leal, R.A. Barbosa, C.R. Oliveira, A.H. Souza, E.H. Valença, M.H. Aguiar-Oliveira, Subjects with isolated GH deficiency due to a null GHRHR mutation eat proportionally more, but healthier than controls, Endocrine 51 (2016) 317–322. [12] M. Kojima, H. Hosoda, Y. Date, M. Nakazato, H. Matsuo, K. Kangawa, Ghrelin is a growth-hormone-releasing acylated peptide from stomach, Nature 402 (1999)
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Effects of growth hormone-releasing hormone gene targeted ablation on ghrelin-induced feeding
MARK
Lucia Recinellaa, Sheila Leonea, Claudio Ferrantea, Annalisa Chiavarolia, Rugia Shohreha, Chiara Di Nisioa, Michele Vaccaa, Giustino Orlandoa,⁎, Roberto Salvatorib, Luigi Brunettia a b
Department of Pharmacy, G. d'Annunzio University, Chieti, Italy Division of Endocrinology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Growth hormone-releasing hormone knockout Ghrelin Feeding NPY AgRP CRH
Impairment of growth hormone (GH) signaling has been associated with increased feeding and adiposity. The gastric hormone ghrelin, in addition to its GH-secretagogue effects, stimulates food intake after both central and peripheral administration. In the present study we further investigated the feeding regulatory role of the ghrelinGH axis in a mouse model of isolated GH deficiency due to targeted ablation of the GH-releasing hormone (GHRH) gene [GHRH knockout (GHRHKO)]. We evaluated the effects of intracerebroventricular ghrelin administration on feeding behavior, related hypothalamic neuropeptides and neurotransmitters, and serum ghrelin levels in mice homozygous for GHRHKO allele (−/−) and heterozygous (+/−) control animals. Vehicletreated GHRHKO mice showed increased food intake compared to heterozygotes, associated with increased circulating ghrelin levels. Moreover, −/− mice showed elevated hypothalamic levels of neuropeptide Y (NPY), agouti-related peptide (AgRP) mRNAs and norepinephrine (NE) and decreased corticotropin-releasing hormone (CRH) mRNA levels. Ghrelin treatment significantly augmented food intake in both genotypes, but the relative increase compared to vehicle-treated animals was higher in −/− than +/− mice. In the hypothalamus, ghrelin increased AgRP and decreased CRH gene expression only in heterozygous mice, while it induced a significant reduction in proopiomelanocortin (POMC) mRNA levels in −/− mice. Ghrelin treatment also decreased hypothalamic serotonin (5-hydroxytriptamine, 5-HT) and dopamine (DA) levels in both genotypes. Additionally, we observed increased DA metabolism induced by ghrelin in both genotypes. In conclusion, dysregulation of the ghrelin-GHRH-GH axis in GHRHKO mice could lead to increased feeding secondary to elevated circulating levels of ghrelin, and the obesogenic phenotype is likely mediated by elevated NPY and AgRP, and decreased CRH gene expression in the hypothalamus.
1. Introduction Besides the well-known somatic growth stimulatory effects, growth hormone (GH) also plays a key role in energy balance [1]. In adipose tissue, GH stimulates lipolysis [2]. Accordingly, GH deficiency (GHD) is characterized by increased fat depots in both humans and mice [3,4]. The effects of GH on feeding have been investigated with inconsistent results [5–9]. We have previously reported that a mouse model of isolated GHD (IGHD) due to targeted ablation of the GH-releasing hormone gene (GHRH knockout, GHRHKO) shows increased food intake, visceral and subcutaneous fat depots and serum adiponectin levels [10]. Similarly, humans with IGHD due to a null mutation in the GHRH receptor gene have higher energy intake (when corrected by body weight) compared to controls [11]. The isolation of the gastric hormone ghrelin as the endogenous ⁎
ligand of the GH secretagogue receptor (GHSR) [12] has provided an intriguing dimension to the role of GH on feeding. GHSR's are expressed in the arcuate nucleus of the hypothalamus (ARC), as well as in other hypothalamic nuclei [13], and GHRH neurons are a major target of ghrelin within the ARC [14,15]. In addition to stimulating GHRH-GH axis, ghrelin has been shown to stimulate feeding and body weight gain when administered either centrally or peripherally [16–18]. The ARC is a major site for transducing peripheral afferents into the neural network involved in feeding control [19]. The ARC includes two populations of peptidergic neurons expressing either neuropeptide Y (NPY)/agouti-related peptide (AgRP) that stimulate food intake, or proopiomelanocortin (POMC)/cocaine- and amphetamine-regulated transcript (CART), which have anorexigenic effects. Both neuronal populations project to neurons located in the lateral hypothalamus (LH) and paraventricular nucleus (PVN), which express orexin and
Corresponding author at: Department of Pharmacy, G. d'Annunzio University, via dei Vestini 31, 66013 Chieti, Italy. E-mail address: [email protected] (G. Orlando).
http://dx.doi.org/10.1016/j.ghir.2017.10.007 Received 1 August 2017; Received in revised form 23 October 2017; Accepted 25 October 2017 1096-6374/ © 2017 Elsevier Ltd. All rights reserved.
Growth Hormone & IGF Research 37 (2017) 40–46
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infection. Immediately after surgery, the mice were injected subcutaneously with 0.5 ml of sterile saline solution and 0.5 ml of 5% glucose solution, and intraperitoneally with amoxicillin/clavulanic acid (30 mg/kg/day). Beginning one week after surgery, at 09:00 a.m., 2.5 month aged −/− (n = 12) and +/− (n = 12) mice were divided into two groups and injected into third ventricle with either ghrelin (3 nmol, 1 μl) or vehicle (saline, 1 μl). Food intake was recorded 1, 2, 8 and 24 h after treatment in each group, by subtracting from supplied pellet the residual in the dispenser and sawdust bedding. Because of the significant difference in total body weight (TBW) between −/− and +/− mice, food intake in grams of chow was normalized to TBW (% of TBW). Finally, animals were euthanized by CO2 inhalation (100% CO2 at a flow rate of 20% of the chamber volume per minute) and blood was collected by cardiac puncture.
corticotropin-releasing hormone (CRH), and drive orexigenic or anorexigenic behavior, respectively [20]. Ghrelin expressing neurons target these hypothalamic peptidergic circuits [21,22], adding to systemicderived ghrelin in modulating feeding control. GHD in GHRHKO animals could possibly involve changes in ghrelin secretion, with consequent effects on feeding control. Hypothalamic aminergic neurotransmitters also play a role in food intake regulation and we have previously reported that adipokines and gut-derived hormones which are known to affect feeding, also modulate hypothalamic dopamine (DA), norepinephrine (NE) and serotonin (5hydroxitriptamine, 5-HT) levels [23–26]. The aim of the present study was to further investigate the roles played by ghrelin in GHRH deficiency-associated dysregulation of feeding. We evaluated food intake, serum ghrelin levels and mRNA levels of CART, CRH, POMC, AgRP, NPY and orexin-A in the hypothalamus, after either vehicle or exogenous ghrelin intracerebroventricular (i.c.v.) administration. We also investigated the effects of exogenous ghrelin on hypothalamic neurotransmitters DA, NE, 5-HT, and their metabolites homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5HIIA) concentrations.
2.3. Serum ghrelin levels Serum total ghrelin levels were measured using the human/mouse/ rat ghrelin enzyme immunoassay (EIA) kit (#K4790-100, Enzo Life Sciences AG, Lausen, Switzerland), following the manufacturer's instruction manual. The sensitivity of the EIA kit was 161 pg/ml. Samples were analyzed in duplicate and the ghrelin levels were determined according to the calibration curves established from standard. The intra and inter-assay coefficients of variation were < 10% and < 15%, respectively.
2. Materials and methods 2.1. Animals and drugs Homozygous mice carrying a targeted ablation of the GHRH gene were previously described [27]. We employed adult (2.5 months old) (10–13 g) homozygous GHRHKO (−/−) male mice (n = 12) and adult (2.5 months old) (20–24 g) male mice heterozygous for the GHRHKO allele (+/−) (n = 12) as control group. GHRHKO offsprings were generated by mating heterozygous males and females, as previously reported [27]. Heterozygous mice were used as control because no significant difference in body weight was observed between +/+ and +/− animals from the 2nd to the 12th week of life [27] despite a modest difference in circulating insulin-like growth factor 1 (IGF1) levels [27]. Indeed, we did not observe any behavioral and neurotransmitter level difference in multiple brain areas between homozygous normal and heterozygous animals (supplementary data). The animals were housed in Plexiglas cages (35 × 20 × 13 cm), and maintained in acclimatized colony rooms (21 ± 1 °C; 55 ± 5% humidity), on a 14 h/10 h light/dark cycle, with continuous ad libitum access to water and food. Mice were fed with a standard rodent chow (Prolab RMH2500, PMI Nutrition International, Brentwood, MO). Housing conditions and experimentation procedures were strictly in agreement with the European Community ethical regulations (EU Directive n. 63/2010) on the care of animals for scientific research, and approved by Italian Health Ministry (Project n. 955/2016-PR). Rat recombinant ghrelin (1 mg/vial) was purchased from Vincibiochem [Vinci (FI), Italy] and diluted in saline.
2.4. RNA extraction, reverse transcription and real-time reverse transcription polymerase chain reaction (real-time RT PCR) Immediately after sacrifice, brains were rapidly removed and individual hypothalami immediately dissected and stored in RNAlater solution (Life Technologies, Carlsbad, CA, USA) at − 20 °C until further processing. Total RNA was extracted from the hypothalami using TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA), as previously reported [28]. One microgram of total RNA extracted from each sample in a 20 μl reaction volume was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's manual. Reactions were incubated in a 2720 Thermal Cycler (Life Technologies, Carlsbad, CA, USA) initially at 25 °C for 10 min, then at 37 °C for 120 min and finally at 85 °C for 5 s. Gene expression was determined by quantitative real-time PCR using TaqMan probe-based chemistry (Life Technologies, Carlsbad, CA, USA). Reactions were performed in MicroAmp Fast Optic 96-well Reaction Plates (Life Technologies, Carlsbad, CA, USA) on an ABI PRISM 7900 HT Fast Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). PCR primers and TaqMan probes were obtained from Life Technologies (Assays-on-Demand Gene Expression Products, Mm00475829_g1 for agouti-related peptide gene, Mm03048253_m1 for neuropeptide Y gene, Mm01964031_s1 for orexin-A gene, Mm00489086_m1 for cocaine- and amphetamine-regulated transcript gene, Mm01293920_s1 for corticotrophin releasing hormone gene, Mm00435874_m1 for proopiomelanocortin gene and Mm00607939_s1 for β-actin gene). β-Actin was used as the housekeeping gene. Data were elaborated with the Sequence Detection System (SDS) software version 2.3 (Life Technologies, Carlsbad, CA, USA). The comparative 2− ΔΔCt method was used to quantify the relative abundance of mRNA and then determine the relative changes in individual gene expression (relative quantification) [29].
2.2. In vivo intracerebroventricular treatment Mice were anesthetized by intraperitoneal injection of ketamine/ xylazine (50 and 5 mg/kg, respectively), and placed in a stereotaxic instrument (David Kopf Instruments, Tujunga, CA). Coordinates for placement of a cannula into the third ventricle were as follows: anteroposterior, + 0.3 mm anterior to the bregma; mediolateral, + 1 mm, right to the bregma; dorsoventral, 3 mm. The site for the cannula was marked. Small holes were drilled into the skull and stainless-steel screws were placed. A hole was drilled through the skull, in the marked site, for the placement of the 24-gauge stainless-steel cannula (1.2 cm long), sterilized in autoclave 2 h before surgery. Once in place, the cannula was attached to the skull using dental cement (Formatray, Salerno, Italy). Sterile obturators were inserted into the cannulas to prevent them from clogging and to reduce the potential for brain
2.5. Hypothalamic neurotransmitter extraction and high performance liquid chromatography (HPLC) determination Immediately after sacrifice, individual hypothalami were rapidly removed and subjected to biogenic amine and metabolite extractive 41
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Fig. 1. Effects of acute i.c.v. ghrelin injection on food intake in ad libitum-fed −/− mice and control +/− animals. Vehicle or ghrelin (3 nmol) was administered by i.c.v. injections, during the light phase, at 09:00 a.m. Food consumption was recorded 24 h after treatment in each group of mice and normalized to total body weight (TBW). Values are given as means ± S.E.M. for each experimental group (n = 6). Vehicle-treated −/− mice consumed more food per TBW than vehicle-treated +/− animals (ANOVA, P < 0.0001; post hoc **P < 0.01 vs. +/− vehicle). Compared to vehicle, ghrelin treatment significantly increased food intake in both genotypes (ANOVA, P < 0.0001; post hoc ***P < 0.001 vs. vehicle-treated gene-matched controls), but the increased feeding response to ghrelin with respect to vehicle-treated animals was higher in −/− than +/− mice (ANOVA, P < 0.0001; post hoc #P < 0.001 vs. +/− ghrelin).
analysis, 1.00 (calibrator sample) was obviously considered the theoretical mean for the comparison. Gaussian distribution of data was assessed by D'Agostino and Pearson omnibus normality test. Differences were considered to be significant when P was < 0.05. As previously reported [30], we calculated the animals randomized for each experimental group on the basis of the “Resource Equation” N = (E + T) / T (10 ≤ E ≤ 20), where E, N and T represent the numbers of degrees of freedom in an ANOVA, animals and treatment, respectively. In our experiments E was 20, for behavioral evaluation. On the other hand, E was 10, for biochemical evaluations. Any sample size, which keeps E between 10 and 20, should be considered as an adequate. If E is < 10 then adding more animals will increase the chance of getting more significant result, but if it is > 20 then adding more animals will not increase the chance of getting significant results [31]. Considering that T value was 4, as regards to behavioral and ghrelin level evaluations, N value was 6, in both genotypes. In agreement with the recognized principles of “Replacement, Reduction and Refinement of Animals in Research”, N value was 3 for neurotransmitter and neuropeptide activity evaluations, requiring different experimental paradigms of extraction and quantification, without adding more animals to the experimental procedures.
procedures [25]. Briefly, samples were treated with 1 ml of 0.05 N perchloric acid containing 0.004% sodium EDTA and 0.010% sodium bisulfite and centrifuged at 4500g for 10 min. The supernatant was filtered on 0.45 μm PTFE sterile filters (Whatman) and directly injected for HPLC. Analyte recovery was satisfactory (≥90%) and reproducible, with percentage relative standard deviation ≤ 10%. The HPLC apparatus consisting of a Jasco (Tokyo, Japan) PU-2080 chromatographic pump and an ESA (Chelmsford, MA, USA) Coulochem III coulometric detector, equipped with microdialysis cell (ESA-5014b) porous graphite working electrode and solid state palladium reference electrode. The analytical cell was set at − 0.150 V, for detector 1 and at +0.300 V, for detector 2, with a range of 100 nA. The chromatograms were monitored at the analytical detector 2. Integration was performed by Jasco Borwin Chromatography software, version 1.5. The chromatographic separation was performed by isocratic elution on Phenomenex Kinetex reverse phase column (C18, 150 mm × 4.6 mm i.d., 2.6 μm). The mobile phase was (10:90, v/v) acetonitrile and 75 mM pH 3.00 phosphate buffer containing octanesulfonic acid 1.8 mM, EDTA 30 μM and triethylamine 0.015% (v/v). Flow rate was 0.6 ml/min and the samples were manually injected through a 20 μl loop. Neurotransmitter and metabolite peaks were identified by comparison with the retention time of pure standard. Neurotransmitter and metabolite concentrations in the samples were calculated by linear regression curve (y = bx + m) obtained with standard. Neither internal nor external standard were necessary for analyte quantification, in the hypothalamus homogenate, and all tests performed for method validation yielded results in accordance to limits indicated in official guidelines for applicability in laboratory trials. The standard stock solutions of DA, NE, 5-HT, HVA, DOPAC and 5-HIIA at 2 mg/ml were prepared in bidistilled water containing 0.004% EDTA and 0.010% sodium bisulfite. The stock solutions were stored at 4 °C. Work solutions (1.25–20.00 ng/ml) were obtained daily progressively diluting stock solutions in mobile phase.
3. Results 3.1. Food intake and serum ghrelin levels As previously reported [10], when food intake was normalized as percentage of TBW, vehicle-treated −/− mice consumed more food than +/− animals (ANOVA, P < 0.0001; post hoc **P < 0.01 vs. +/− vehicle). Ghrelin treatment significantly augmented food intake both in −/− and +/− mice 24 h following injection (ANOVA, P < 0.0001; post hoc ***P < 0.001 vs. vehicle-treated gene-matched controls), and the relative increase compared to vehicle-treated animals was higher in −/− than +/− mice (ANOVA, P < 0.0001; post hoc #P < 0.001 vs. +/− ghrelin) (Fig. 1). Conversely, no change in feeding was induced by ghrelin 1, 2 and 8 h post injection [data not shown]. As compared to +/− animals, serum ghrelin levels were higher in vehicle-treated −/− mice. In addition, ghrelin administration resulted in increased serum levels of ghrelin in both genotypes [(+/− vehicle 4.45 ± 0.15 ng/ml; −/− vehicle 6.15 ± 0.28**ng/ ml; +/− ghrelin 20.75 ± 1.23***ng/ml; −/− ghrelin
2.6. Statistical analysis Statistical analysis was performed using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, CA, USA). All data were collected from each of the 24 animals used in the experimental procedure and means ± S.E.M. were collected for each experimental group and analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. As for gene expression 42
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Fig. 2. Relative gene expression of hypothalamic agouti-related peptide (AgRP) (A), neuropeptide Y (NPY) (B), orexin-A (C), cocaine- and amphetamine-regulated transcript (CART) peptide (D), corticotropin-releasing hormone (CRH) (E) and proopiomelanocortin (POMC) (F), 24 h after treatment with either vehicle or ghrelin in −/− mice and control +/− animals, as determined by real-time RT PCR. Data were calculated using the 2− ΔΔCt method; they were normalized to β-actin mRNA levels and then expressed as relative to vehicle-treated +/− mice (calibrator sample, defined as 1.00). Values are given as means ± S.E.M. for each experimental group. Vehicle-treated −/− mice had higher AgRP (ANOVA, P < 0.0005; post hoc ***P < 0.001 vs. +/− vehicle) (A) and NPY (ANOVA, P < 0.0001; post hoc ***P < 0.001 vs. +/− vehicle) (B) and lower CRH (ANOVA, P < 0.05; post hoc *P < 0.05 vs. +/− vehicle) (F) gene expression than +/− animals. Ghrelin treatment decreased POMC mRNA levels (ANOVA, P < 0.001; post hoc ***P < 0.001 vs. −/− vehicle) (E) in −/− mice with respect to vehicle. On the other hand, ghrelin increased AgRP (ANOVA, P < 0.001; post hoc *P < 0.05 vs. +/− vehicle) (A) and decreased CRH (ANOVA, P < 0.05; post hoc *P < 0.05 vs. +/− vehicle) (F) gene expression in +/− mice.
23.68 ± 1.45***ng/ml) ANOVA, P < 0.0001; post hoc **P < 0.01 vs. +/− vehicle, ***P < 0.001 vs. vehicle-treated gene-matched controls].
***P < 0.001 vs. +/− vehicle) (Fig. 2A) and NPY (ANOVA, P < 0.0001; post hoc ***P < 0.001 vs. +/− vehicle) (Fig. 2B), and decreased CRH (ANOVA, P < 0.05; post hoc *P < 0.05 vs. +/− vehicle) (Fig. 2F) gene expression levels. No genotype-related differences were observed in orexin-A, CART and POMC mRNA levels (Fig. 2C–E). Ghrelin treatment, while stimulating food intake in both genotypes, differently affected hypothalamic peptide gene expression. In −/− mice, ghrelin induced a significant reduction in POMC mRNA levels
3.2. Hypothalamic peptide gene expression As compared to vehicle-treated +/− animals, vehicle-treated −/− mice had increased AgRP (ANOVA, P < 0.0005; post hoc 43
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A
C
B
Fig. 3. Effects of acute i.c.v. ghrelin injection in ad libitum-fed −/− mice and control +/− animals on hypothalamic monoamines (ng/mg wet tissue). Dopamine (DA) (A), norepinephrine (NE) (B) and serotonin (5-hydrohytryptamine, 5-HT) (C) levels are expressed as mean ± S.E.M. for each experimental group. Compared to vehicle-treated +/− animals, −/− vehicle-treated mice showed higher NE levels (ANOVA, P < 0.05; post hoc *P < 0.05 vs. +/− vehicle) than their respective +/− littermates (B). Compared to vehicle, ghrelin treatment decreased DA (ANOVA, P < 0.001; post hoc **P < 0.01 vs. vehicle-treated gene-matched controls) (A) and 5-HT levels (ANOVA, P < 0.01; post hoc *P < 0.05 vs. vehicletreated gene-matched controls) (C) in both genotypes leaving unaffected NE levels (ANOVA, P < 0.05; post hoc #P < 0.05 vs. +/− ghrelin) (B).
detected on 5-HIIA/5-HT ratio values (+/− vehicle 1.05 ± 0.11; −/− vehicle 1.043 ± 0.02; +/− ghrelin 1.01 ± 0.08; −/− ghrelin 0.97 ± 0.20).
compared to vehicle (ANOVA, P < 0.001; post hoc ***P < 0.001 vs. −/− vehicle) (Fig. 2E), while it did not affect the other neuropeptides. On the other hand, in +/− mice, ghrelin administration increased AgRP (ANOVA, P < 0.001; post hoc *P < 0.05 vs. +/− vehicle) (Fig. 2A) and decreased CRH (ANOVA, P < 0.05; post hoc *P < 0.05 vs. +/− vehicle) (Fig. 2F) gene expression, leaving unaffected the other neuropeptides.
4. Discussion In confirming our previous findings showing that GHRHKO mice have increased food intake compared to +/− animals (Fig. 1), the present work demonstrates that GHRH homozygous gene deletion leads to increased circulating ghrelin levels and a characteristic pattern of neuropeptide and neurotransmitter signaling in the hypothalamus. The control of pituitary GH secretion by ghrelin is finely tuned by negative feedback regulatory loops. Decreased circulating ghrelin levels have been shown in acromegalic patients compared to healthy subjects [32,33], and after administration of exogenous GH, while plasma ghrelin is increased by hypophysectomy, in rats [34]. Accordingly, we hypothesized that in GHRHKO mice ghrelin levels could be upregulated due to decreased inhibitory feedback by GH and IGF1, and that this could play a role in increasing feeding and adipose tissue stores, possibly through modulation of neuropeptides and neurotransmitters in the hypothalamus. Our results show that GHRHKO mice have higher serum ghrelin levels, underlying their increased food intake as compared to heterozygous mice. Exogenous ghrelin administration further increase feeding in both genotypes, with a higher incremental effect in GHRHKO animals (Fig. 1). Our data also show that the increased feeding phenotype by GHRHKO mice could be related to specific involvement of neural pathways in the hypothalamus, which are also affected by
3.3. Hypothalamic aminergic neurotransmitters and metabolites Compared to vehicle-treated heterozygous controls, −/− vehicletreated mice had higher NE concentrations, in the hypothalamus (ANOVA, P < 0.05; post hoc *P < 0.05 vs. +/− vehicle) (Fig. 3B) while no genotype-related differences were observed in DA and 5-HT levels. Ghrelin treatment decreased DA (ANOVA, P < 0.001; post hoc **P < 0.01 vs. vehicle-treated gene-matched controls) (Fig. 3A) and 5HT levels (ANOVA, P < 0.01; post hoc *P < 0.05 vs. vehicle-treated gene-matched controls) (Fig. 3C) in both genotypes, leaving unaffected NE levels. Additionally, we did not observe any difference between vehicletreated +/− and vehicle-treated −/− mice in DA and 5-HT metabolism, evaluated as HVA/DOPAC and 5-HIIA/5-HT ratio values, respectively. Compared to vehicle administration, ghrelin increased HVA/ DOPAC ratio values, in both genotypes [(+/− vehicle 0.74 ± 0.03; −/− vehicle 0.61 ± 0.05; +/− ghrelin 2.38 ± 0.06**; −/− ghrelin 2.12 ± 0.10**) ANOVA, P < 0.0001; post hoc **P < 0.01 vs. vehicle-treated gene-matched controls]. Finally, no effects were 44
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pathways, in the hypothalamus [49]. The existence of a negative feedback system linking brain serotonin levels to plasma ghrelin in the regulation of energy homeostasis in mice has been suggested [44]. Pretreatment with 5-HT injected into the PVN was found to inhibit feeding stimulated by ghrelin injected into the PVN [45]. We did not find any significant genotype-related difference in vehicle-treated mice in hypothalamic 5-HT levels and 5-HIIA/5-HT ratio. Similarly, ghrelin injection reduced hypothalamic 5-HT levels (Fig. 3C), without any effect on 5-HT metabolism, evaluated as 5-HIIA/ 5-HT ratio, to a similar degree in both genotypes. Considering the inhibitory role of 5-HT on feeding, our findings showing decreased 5-HT levels after ghrelin treatment could be related to the orexigenic effects of ghrelin in both genotypes. In conclusion, dysregulation of the ghrelin-GHRH-GH axis in GHRHKO mice leads to increased feeding, likely caused by increased circulating ghrelin levels. The obesogenic phenotype could be mediated by elevated NPY and AgRP, and decreased CRH gene expression, together with increased NE levels in the hypothalamus.
exogenous ghrelin administration. Our experiments show that vehicle-treated GHRH −/− mice have a significant higher increase in NPY and AgRP (Fig. 2A–B), and decrease in CRH (Fig. 2F) mRNAs levels in the hypothalamus when compared to +/− animals. Considering that NPY and AgRP play a feeding stimulatory role, while CRH reduces feeding, their reciprocal modulation in −/− animals could be related to their increased endogenous ghrelin levels as compared to +/− controls. Ghrelin has been shown to stimulate feeding in wild type animals, possibly through increased NPY and AgRP levels [16,17,35,37,38]; moreover, feeding stimulation by ghrelin is lost in AgRP and NPY double KO mice [36]. Additionally, electrophysiological approaches have shown that ghrelin can activate NPY and AgRP co-expressing neurons and simultaneously reduce the activity of POMC neurons [21]. Our findings further show that exogenous ghrelin administration stimulates AgRP and decreases CRH gene expression but only in heterozygous and not in −/− mice. The latter genotype is characterized by basally increased AgRP and decreased CRH mRNA levels (as seen in vehicle-treated animals), possibly due to increased endogenous ghrelin concentrations, and this may explain why these transmitters cannot be further modified by exogenous ghrelin administration. On the other hand, the higher feeding response to ghrelin shown by −/− animals could be related to the decrease in POMC gene expression that we have observed being induced by ghrelin in −/− animals but not in heterozygotes. The role of POMC-derived peptides is somewhat controversial, since both the orexigenic β-endorphin and the anorexigenic α-MSH could be processed from POMC gene transcript [19]. Previous studies have hypothesized that increased feeding by ghrelin could be also mediated by decreased melanocortin signaling through central upregulation of α-MSH degrading enzyme prolyl carboxypeptidase [39]. Furthermore, NPY and AgRP co-expressing neurons stimulated by ghrelin could exert unidirectional inhibitory tone on POMC neurons [40,41]. We also found increased NE levels in vehicle-treated −/− mice compared to +/− controls (Fig. 3B). The role of NE as a central transducer of appetite/satiety signals is controversial, with either inhibiting or stimulating effects, possibly related to the activation of α1or α2-adrenoceptors, respectively [42]. The anorectic effects of central and peripheral leptin administration have been linked to a significant reduction in hypothalamic NE concentration in several hypothalamic nuclei, such as ARC, PVN and dorsomedial nucleus in the rat [43]. Moreover, elevated circulating levels of NE were found in ob/ob leptindeficient mice, suggesting an inverse correlation between NE and leptin as one of the mechanisms of feeding regulation by this peptide [20]. We have previously found that omentin-1, a fat depot-specific secretory protein stimulating feeding in the rat, also increases NE synthesis and release, in the hypothalamus [25]. Consequently, increased feeding behavior observed in vehicle-treated −/− mice could also be driven by augmented NE levels in the hypothalamus. However, ghrelin injection did not modify hypothalamic NE levels, compared to vehicle-treated gene-matched controls. The role of DA in feeding control is still unsettled, with studies showing both inhibitory and stimulatory effects. On one side, DA stimulates feeding after its injection into the lateral hypothalamus [46]. On the other hand, DA administration into the perifornical hypothalamus produces anorexigenic effects [47], and increased DA transmission is associated with the anorectic effects of amphetamines [48]. Our findings show no difference on hypothalamic DA levels in vehicletreated −/− mice compared to +/− controls. In addition, ghrelin administration decreases DA in both strains of mice. We also found that ghrelin treatment is associated to increased HVA/DOPAC ratio, which could be related to increased DA metabolism induced by the peptide. On the other hand, we did not observe any direct influence by the peptide on hypothalamic DA release, in vitro [23]. Nevertheless, our findings of increased AgRP and reduced POMC gene expression in +/− and −/− mice, respectively, following ghrelin injection further support a possible interaction between melanocortin and dopaminergic
Conflict of interest statement The authors declare no conflict interest. Funding This work was supported by grants from the Italian Ministry of University (FAR 2016). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ghir.2017.10.007. References [1] D. Le Roith, C. Bondy, S. Yakar, J.L. Liu, A. Butler, The somatomedin hypothesis: 2001, Endocr. Rev. 22 (2001) 53–74. [2] H.M. Goodman, Y. Schwartz, L.R. Tai, E. Gorin, Actions of growth hormone on adipose tissue: possible involvement of autocrine or paracrine factors, Acta Paediatr. Scand. Suppl. 367 (1990) 132–136. [3] M. Alba, D. Fintini, R. Salvatori, Effects of recombinant mouse growth hormone treatment on growth and body composition in GHRH knockout mice, Growth Hormon. IGF Res. 15 (2005) 275–282. [4] E. Gomes-Santos, R. Salvatori, T.O. Ferrão, C.R. Oliveira, R.D. Diniz, J.A. Santana, F.A. Pereira, R.A. Barbosa, A.H. Souza, E.V. Melo, C.C. Epitácio-Pereira, A.A. Oliveira-Santos, I.A. Oliveira, J.A. Machado, F.J. Santana-Júnior, J.A. BarretoFilho, M.H. Aguiar-Oliveira, Increased visceral adiposity and cortisol to cortisone ratio in adults with congenital lifetime isolated GH deficiency, J. Clin. Endocrinol. Metab. 99 (2014) 3285–3289. [5] M.J. Azain, T.J. Roberts, R.J. Martin, T.R. Kasser, Comparison of daily versus continuous administration of somatotropin on growth rate, feed intake, and body composition in intact female rats, J. Anim. Sci. 73 (1995) 1019–1029. [6] M. Bohlooly-Y, B. Olsson, C.E. Bruder, D. Lindén, K. Sjögren, M. Bjursell, E. Egecioglu, L. Svensson, P. Brodin, J.C. Waterton, O.G. Isaksson, F. Sundler, B. Ahrén, C. Ohlsson, J. Oscarsson, J. Törnell, Growth hormone overexpression in the central nervous system results in hyperphagia-induced obesity associated with insulin resistance and dyslipidemia, Diabetes 54 (2005) 51–62. [7] J. Klindt, J.T. Yen, F.C. Buonomo, A.J. Roberts, T. Wise, Growth, body composition, and endocrine responses to chronic administration of insulin-like growth factor I and(or) porcine growth hormone in pigs, J. Anim. Sci. 76 (1998) 2368–2381. [8] K. Malmlöf, N. Din, T. Johansen, S.B. Pedersen, Growth hormone affects both adiposity and voluntary food intake in old and obese female rats, Eur. J. Endocrinol. 146 (2002) 121–128. [9] T.J. Roberts, M.J. Azain, Somatotropin treatment reduces energy intake without altering protein intake in pigs selecting between high and low protein diets, J. Nutr. 127 (1997) 2047–2053. [10] L. Recinella, R. Shohreh, R. Salvatori, G. Orlando, M. Vacca, L. Brunetti, Effects of isolated GH deficiency on adipose tissue, feeding and adipokines in mice, Growth Hormon. IGF Res. 23 (2013) 237–242. [11] A.A. Oliveira-Santos, R. Salvatori, E. Gomes-Santos, J.A. Santana, Â.C. Leal, R.A. Barbosa, C.R. Oliveira, A.H. Souza, E.H. Valença, M.H. Aguiar-Oliveira, Subjects with isolated GH deficiency due to a null GHRHR mutation eat proportionally more, but healthier than controls, Endocrine 51 (2016) 317–322. [12] M. Kojima, H. Hosoda, Y. Date, M. Nakazato, H. Matsuo, K. Kangawa, Ghrelin is a growth-hormone-releasing acylated peptide from stomach, Nature 402 (1999)
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