Revista Bionatura
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Biological   activity of a new Growth hormone secretagogue: study in fish and murine cell   line
La   actividad biológica de un nuevo secretagogo de la hormona de crecimiento:   estudio en el pescado y la línea celular murina

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Rebeca Martínez,   Katerina Gonzalez, Alain Gonzalez, Kenia Ubieta,   Fidel Herrera, Osvaldo Reyes, Hilda Garay,   Ayme Oliva, Elsa Rodriguez, Mario Pablo Estrada


Growth hormone   (GH) has pleiotropic functions in all vertebrates. In addition to its essential   role in the regulation of body growth and development, it can also influence   reproduction, immunity, osmoregulation, and behavior Immune and neuroendocrine   systems have bidirectional communications and it is well document the enhancing   action of GH on teleost immune system .It is desirable controlled GH administration   to allow growth and stimulation of the innate immune system of fish. In this   study, we have characterized a chemical peptide compound, A228, designed by   molecular modeling, which is able to perform the function of a GH peptide secretagogue. In pituitary cell culture, the peptide A228 induces GH secretion. It is able to increase superoxide production in tilapia peripheral blood leukocytes cultures  and in a macrophage cell line J774 from mice, therefore using this molecule,  innate immune system stimulation is obtained in vitro both in fish and in mammals  cell cultures. In this paper is also shown the biological action in vivo of   the molecule, to assess growth stimulation in tilapia larvae.
Keywords   : Biological activity, Growth hormone secretagogue, murine cell line.

La hormona del   crecimiento (GH) tiene funciones pleiotrópicas en todos los vertebrados y juega un papel esencial en la regulación del crecimiento y el desarrollo del cuerpo. También puede influir en la reproducción,  la inmunidad, la osmorregulación, y el comportamiento inmunológico. Los sistemas neuroendocrinos tienen comunicaciones bidireccionales y esta bien  documentada la acción de la GH sobre el sistema inmune de  teleósteo. La administración  controlada de GH permitir el crecimiento y la estimulación del sistema inmune innato de los peces. En este estudio, hemos caracterizado un compuesto peptídico químico, A228, diseñado  por modelado molecular, que es capaz de realizar la función de un péptido  secretagogo de GH. En el cultivo de células pituitaria, el péptido A228 induce la secreción de GH y es capaz de aumentar la producción de superóxido en tilapia.  En este documento también se muestra la acción biológica in vivo de la molécula,   para evaluar la estimulación del crecimiento en larvas de tilapia.
Palabras clave: actividad biológica, scretagogo, hormona del crecimiento, línea   celular murina.

Growth hormone   (GH) is a pluripotent hormone with essential role in the regulation of body   growth and development; it can also influence reproduction, immunity, osmoregulation,  and behavior (1).It is produced by the pituitary gland in teleosts as in other   vertebrates .GH gene is also expressed in other tissues of fish, especially  in lymphoid organs and cells (2). The expression of GH is tightly regulated   by several factors.
There are have   been demonstrated the interactions among elements of the endocrine and immune   systems in fish. And it is desirable controlled GH administration to allow growth   and stimulation of the innate immune system of fish.
The innate response   is the basis of the immune defence of invertebrates and lower vertebrates. In   fish, the innate immune response has been considered the essential component   in combating pathogen invasions due to the limits placed on their adaptive immune   response.(3) The macrophage cell lineage represents an important group of cells which play a central role in the initiation and coordination of the immune response    
Among the different   peptides involved in the regulation of expression of GH are the GHS. The synthetic   GH secretagogues (GHSs) consist of a family of ligands, first described by Momany   et al. 1981(4). Multiple efforts have been directed toward finding the active   molecules, yielding the discovery of new GHSs (5). GHS bind to a receptor inducing   calcium mobilization, named GHS receptor (GHSR) first identified in pigs and   humans. but currently GHS receptor (GHSR) has been identified in teleosts and   birds (6,7,8,9).The ligand endogenous of this receptor correspond to the ghrelin   hormone , which elicits a diverse biological action besides increasing GH secretion   from the anterior pituitary gland, enhancing anabolic effects such as increasing   appetite, adiposity and blood glucose, enhancing gastric function and increasing   cardiac output via GHS-R. GHS-R is expressed predominantly in the brain and   pituitary, but it is also expressed in many peripheral organs such as the heart,   lungs, liver, kidneys, pancreas, stomach, small and large intestines , adipose   tissue and immune cells
The moleculeA228   was described by the exhaustive molecular modeling of the human GRLN receptor   using bioinformatics tools, after which a virtual library was built and a massive   docking experiment was performed against the receptor model (10).
Previous results   have demonstrated the biological activity of another GHS , the peptide A233   , that induces GH secretion and it is also able to increase superoxide production   in tilapia head-kidney leukocyte cultures. This effect is blocked by preincubation   with the GHS receptor antagonist [D-Lys3]- GHRP6.and GH immunonetralization   experiments propose a GH-mediated mechanism for the action of A233. The in vivo   biological action of the decapeptide was also demonstrated for growth stimulation   in fish larvae and the enhancement of some parameters of innate immune system   in the treated larvae.
The aim of this   study was to assess the biological activity of another GHS, the synthetic peptide   A228 as a stimulator of growth and the innate immune system of teleosts fish,   through studies performed in vitro and in vivo. It is also interesting to know   if in a murine macrophage cell line J774 , the A 228 could stimulate the superoxide   anion production .
Tilapia (Oreochromis   sp.) juvenile and larvae were obtained from the Center for Aquaculture of Mamposto´n    
(CPAM). Fish were   kept alive in aerated freshwater under a 12 h light:12 h darkness photoperiod.   They were fed commercial dry diet for fish (CENPALAB, Habana, Cuba).Water temperature   was maintained at 26 and 28 8C. All animal experiments were previously approved   by the Ethics Committee of the Center for Genetic Engineering and Biotechnology,   Havana, Cuba.
GH secretagogues   
The decapeptide   A228 (GKFGDLSPEHQ) y A233 (GKFDLSPEHQ) with an internal   lactam bond between side chains of underlined amino acids) was manually synthesized   on a solid-phase support. Crude peptide was purified by reverse-phase, high-performance,   liquid chromatography up to 95% on a C-18 preparative column with an acetonitrile/water   linear gradient. Trifluoracetic acid was used in both solvents for counter-ion   pair formation. The correct sequence of the purified peptide was confirmed by   electrospray mass spectrometry (Micromass, Manchester, UK). The positive control   used was GHRP6 (Lipotec, Barcelona, Spain) lyophilized peptides were reconstituted   in PBS.
Primary culture   of pituitary cells
The in vitro effects   of A233 GHS were examined using cells dispersed from whole pituitary. Mature   tilapias of both sexes weighing 300-500 g were used after anesthesia in tricaine   methanesulfonate (MS-222, Sigma). Pituitaries were collected aseptically in   isotonic medium (Krebs bicarbonate- Ringer solution, 330 m Osmolal, pH 7.4)   supplemented with penicillin (100 IU/ml), streptomycin (0.1 mg/ml), and nystatin   (250 IU/ml, all from Sigma). The pituitaries were diced with a sterile razor   blade and treated with collagenase for 1 h at room temperature in 2.5 ml trypsin-EDTA   solution (0.25% trypsinC0.02% EDTA in PBS, pH 7.4). Tissues were
aspirated repeatedly   through a pipette during enzymatic treatment to promote dissociation of cells.   The process was terminated by the addition of 0.5 ml (20%) fetal bovine serum   (Sigma). Cells were counted on a hemocytometer under a light microscope and   viability determined by trypan blue exclusion. Viability of the cells was always   O95%. Cells were then plated at a density of 4.0!105 cells/well into a 24-well   plate (Falcon, Primaria 24, Becton Dickinson, Franklin Lakes, NJ, USA) at a   volume of 300 ml/well of isotonic medium supplemented with 10% fetal bovine   serum. The cells were preincubated for 4 days at 26-28 8C under a humidified   atmosphere of 95% O2 and 5% CO2, with one change of culture medium at 48 h post-plating.   Before each experiment, cells were washed once with serum-free medium. A final   300 ml serum-free medium was added containing A233, GHRP6 (Lipotec), or control   medium without hormones. The medium was replaced at 4 h.Incubations were terminated   at 8 h, and hormone release was quantified for the 0-4 and 4-8 h intervals.   GH release was expressed as secretion per unit volume of medium (ng/ml).
GH assays   
The GH secreted   in vitro was measured using a noncompetitive ELISA as described by Lugo et al.   2008 (11). The 96-well MaxiSorp plates (Nalge Nunc International, Roskilde,   Denmark) were coated (3 h at 37 8C) with anti-tilapia GH monoclonal antibody   1 (tiGH1 mAb; CIGB, Santi Spiritus, Cuba) at 10 mg/ml in 0.05 M carbonate buffer   (pH 9.6, 100 ml/well). The plates were washed two times with PBS-T (137 mM NaCl,   2.7 mM KCl, 4.3 mM Na2HPO4 .7H2O, and 0.05% Tween 20, pH 7.3) and blocked with   3% skim milk (Oxoid, Cambridge, UK) in PBS 1! (200 ml/well) for 1 h at 37 8C.   A standard tiGH curve in the range of 35-0.136 ng/ml was obtained by twofold   dilutions in 0.5% skim milk in PBS 1!, dispensed by duplicate in the same plate.   Test samples were diluted at a ratio of 1:2 as described earlier and were incubated   on the plates overnight at 4 8C. After washing the plates four times with PBS-T,   100 ml HRP - tiGH2 mAb conjugate (CIGB), diluted at a ratio of 1:15 000 in PBS   1!containing 0.5% skim milk, were added to each well. The plates were incubated   for 1 h at 37 8C and then washed eight times with PBS-T. Then, the substrate   buffer (0.2 M Na2HPO4, 0.1 M citric acid (pH 5.0) containing 0.5 mg/ml ortho-phenylenediamine   and 5 ml 30% H2O2) was added (100 ml/well). The reaction was stopped 15 min   later by adding 50 ml of 2.5 M sulfuric acid per well. The absorbance was measured   at 492 nm using the Titertek Multiskan Plus spectrophotometer. The accepted   upper limit of the assay background was 0.094. The lower detection limit of   the assay was 0.1 ng/ml. The degree of variation and intra- and interassay coefficients   of variation are 3.90 and 13.45% respectively.
Isolation and   culture of leukocytes
Peripheral blood   leukocytes were isolated as described by Acosta et al, 2010(12) .Tilapias were   anesthetized in tricaine methanesulfonate (MS222), and blood was collected from   the caudal vessels using a heparinized syringe. Blood was diluted 1:2 in RPMI-1640   medium, containing 4 μg/mL of gentamicin. The mixture was placed on Ficoll-Paque™   Research Grade (Amersham Pharmacia Biotech, Sweden) and centrifuged at 400 g   for 25 min. The leukocyte band was harvested, washed with phosphate-buffered   saline (PBS, pH 7.6), and suspended in RPMI-1640 medium with 0.5% tilapia serum,   and cultured at 25 _C, in 5% CO2 for 24 h. Leukocyte viability was assessed   by trypan blue exclusion (viability N95%).
Growth conditions   for macrophage cell line J774
Murine macrophage   cell lines J774 was obtained from the American Type Culture Collection (ATCC   ). The cells were cultured as monolayers in Dulbecco's Modified Essential Medium   (DMEM) F12 supplemented with 20% fetal bovine serum,100 mg/mL penicillin/streptomycin,   and 2mM L-glutamine (Mediatech, Inc., Herndon, VA) at 37 1C in a humidified   atmosphere of 5% CO2. The cultured cells were grown to 70-80% confluence in   75 cm2 flasks for preparation of samples that were used in further analysis.    
Superoxide   production in cell culture
To analyze the   effect of GHSs on the immune system, the superoxide anion was measured in tilapia   PBLs and macrophage cell line J774 as the reduction of NBT (Sigma) as described   by Yada et al. 2006 (13)
Growth performance   experiment
The growth promoting   activity of A233 was evaluated in tilapia larvae (Oreochromis sp.) of 0.007G0.001   g mean weight and (n=200). The effect was compared with GHRP6 as positive control.   The fish were acclimated at
28 8C in 120 l   tanks with running fresh water for 1 week before the experiment. They were fed   with a basal diet to satiation twice a day. Before treatment, the tanks were   cleaned. The fish were immersed into each treatment for 60 min without water   recirculation. The immersion procedure was performed three times a week, for   a period
of 1 month. Three   experimental groups were handled with the following treatments: groups 1 and   2 received the peptides A228 and GHRP6 (positive control) at a dose of 0.1 mg/l,   respectively, and a no treated group in the third one. Growth-promoting effects   were evaluated by measuring the body weight increase.
Statistical   analysis
Results were evaluated   using GraphPad Prism version 4.0 for Windows, GraphPad Software, San Diego,   CA, USA. All results are expressed as the mean ±S.D. Statistical analysis   was performed using one-way ANOVA by Newman-Keuls's or Dunnett's method for   data with normal distribution and equal variances: GH assays, superoxide production,   growth performance experiment of tilapia (Oreochromis sp.) larvae. Data with   unequal variances were analyzed by the Kruskal-Wallis test followed by Dunn's   multiple comparisons post-test. Treatments were considered to be significantly   different if P<0.05.
The molecules   A 228 and A233 (Fig. 1) were selected from the virtual libraries   described by Rodriguez et al. 2007(10), and, in order to determine in vitro   and in vivo whether this novels mimetic molecules function as GHSs, the following   assays were conducted on the growth and immune system.

GH in vitro   assays using pituitary cell culture
To evaluate the   effect of A228 and A233 peptide on GH secretion, we performed an in vitro culture   of cells in the pituitary gland of tilapia (Oreochromis sp.). the peptides   at a concentration of 10 nM stimulated GH secretion at 8 h by cells in the anterior   pituitary gland (Table). The stimulatory effect of GRLN and   GHSs on the release of GH in vitro by the pituitary gland has been reported   in mammals, birds, and different species of fish (14,15,16 17, 18).

Determination   and characterization of superoxide anion induction in PBLs and macrophage cell   line J774
Superoxide anion   produced by cultured tilapia (Oreochromis sp.) PBLs were measured by the addition   of A 228 as positive control GHRP-6 was included and also A233, which in previous   results was capable of enhance superoxide anion production in leukocytes from   tilapia head kidney. The peptides induced significant increases in superoxide   production. The concentration of peptide used was 10 n M taking in consideration   previous results ( 19) , The increase of superoxide anion production was statistically   higher in cells stimulated with the peptides, to compare with no treated cells,   among the different treatments there were not statistical differences, in both   cultures, PBLs ( Fig. 2 ) this effect is similar to that reported   by Acosta et al. 2010,(12) who stimulated phagocytic peripheral blood cells   of tilapia (Oreochromis sp.) with recombinant tiGH (Oreochromis hornorum).    

The results obtained   in the production of superoxide anion with the macrophage cell line J774.after   the stimulation with the peptides, were very similar, there were obtained statistically   differences in the superoxide anion production of cells treated with the peptides   , and compare with negative control. (Fig. 3)

In vivo biological   activity assays
Experiments were   performed to evaluate the biological effect of the peptide on the growth rate   of tilapia larvae. The effect of these peptides on fish growth was determined   by measuring the increase in body weight. The tilapia larvae treated with A228   showed a significant increase in body weight compared with the controls as well   as the larvae treated with GHRP -6 , just 9 days after treatment.
In this study,   we demonstrate the effect of another peptide molecule having internal cycles   and composed solely of L-amino acids that are capable of exerting, due to their   chemical structure, similar functions to those attributed to GRLN, des-acyl   GRLN, and other peptidic GHS. They are a group of peptide compounds and peptides   capable of stimulating production and GH secretion in vitro and in vivo.
There were not   differences between both peptides A228 and A233, the structural difference do   not interfere on GH secretagogue function at least in this kind of primary culture   of pituitary cells experiment.
Our results are   similar to those obtained in tilapia (Oreochromis mossambicus), where   the effect of GRLN on GH secretion in vitro was dependent on the concentration   of the endogenous secretagogue used (15). Other researchers have reported the   stimulatory effect of the synthetic peptides GHRP-6, PACAP, and PACAP-related   peptide from Clarias gariepinus on the release of GH in vitro by the   pituitary gland of tilapia (Oreochromis niloticus); (11,19).There should   be conducted an experiment using the A228 peptide to study the GH releasing   in vivo after the administration of GHS.
It has been reported   that the immune system of both fish and mammals possesses both non-specific   and specific immune responses with cellular and humoral components. However,   fish depends more heavily on non-specific defence mechanisms. The use of immunostimulants   given as dietary supplement can improve the innate defense of aquatic animals   principally fish and thus providing resistance to pathogens during period of   stress, such as grading, sea transfer and vaccination.
Neutrophils and   macrophages are known as the phagocytic leucocytes in fish peripheral blood   (20) . Macrophages play a key role in the host immune system. They are in the   first line of defence, participating in detection and identification of potential   pathogens. As part of mechanisms involved in the innate system they respond   to stimuli activating phagocytosis and releasing reactive oxygen and nitrogen   species which destroy microbes. They also initiate the inflammatory response   through cytokine production, and furthermore, macrophage acts a link between   the innate and adaptive immune responses acting as antigen-presenting cells   to prime T cells. ( 21)
The existence   of the binding sites of GH has been shown in both types of leucocytes in the   gilthead sea bream Sparus aurata .It is known also that the administration   in vivo and in vitro GH stimulates the production of superoxide anion after   phagocytosis as a mechanism for elimination of pathogens (2)
In our result   we could state the biological action of or peptide in fish and mammals system,   There are controversial reports about the effects of ghrelin on phagocytotic   activity of leukocytes. In fish (rainbow trout), the administration of ghrelin   increased phagocytosis and superoxide production in zymosan-stimulated leukocytes,   which was abolished by pretreatment of leukocytes with a GHS-R antagonist (2).   It was also shown that ghrelin increased mRNA levels of superoxide dismutase   and GH in leukocytes, suggesting that the effects of ghrelin was mediated, at   least in part, by stimulating GH secretion from leukocytes. On the other hand,   ghrelin administration reportedly reduced the elevated phagocytic activity of   peritoneal macrophages induced by acute cold-restraint stress in rats ( 22 )   . In our study we have found an increase of superoxide anion in fish PBL and   cell line J774 from murine macrophages after A228 stimulation even has been   reported that ghrelin modulates phagocytosis directly or indirectly via GH,   but in a different way in different species it should be considered the treatment   conditions to evaluate this biological activity.
GHSs are useful   molecules as growth enhancement molecules These synthetic molecules are effective   in stimulating production and release of endogenous hormone as a physiological   response, with no side effects on the pituitary or toxicity potential; besides   their low molecular weight, it makes a better entrance to the organism.
In our laboratory,   we have successfully employed the immersion bath technique to study the effects   of nutritional supplements and growth factors on growth control and the immune   system in fish (12). The immersion bath method used for our studies requires   little manipulation and causes minimum stress to fish during treatment. There   is evidence suggesting that the gill pillar cells are a possible entry site   for some molecules when fish are treated by immersion bath (6).
This study evaluated   the biological function of synthetic peptide A228 on weight gain of tilapia    (Oreochromis sp). Tilapia larvae showed a significant increase in growth   after 9 days of treatment with peptide A228 (0.1 mg/l). All animals received   the same commercial diet, so the increase in weight and height is due to the   administration of peptide A228. The positive control group represented by the   fish treated with the peptide GHRP6 significantly increased their growth. Similar   results were obtained in a former work to administrate A233 peptide, another   GHS, to fish larvae enhancing growth and some. humoral innate immune system   parameters (Fig. 4)(19).

These results   are similar to those found in mice where there was an increase in body weight   after dosing by s.c. injection of the synthetic peptide GHRP2 (23). In adult   rats, GHRP6 also increased body weight (24) as well as other GHSR agonists,   like SM-130686 administered orally (25) and BIM-28131 administered by s.c. injection   (26). It has been reported that GHSR1a agonist, a pentapeptide with D-amino   acids, promotes weight gain in rats, by i.p. administration during 7 days (27).   Moreover, the administration, by the same method, of GH tilapia (O. hornorum)   secreted into the culture supernatant of yeast Pichia pastori (28) to larvae   of tilapia (Oreochromis sp.), significantly increased growth of these animals.   Administration of tilapia recombinant neuropeptide Y (Oreochromis sp.) to larvae   of African catfish (C. gariepinus) also produced an increase in animal weight   (29).
In addition, treatment   with PACAP and PACAP-related peptide from C. gariepinus larvae of African catfish   (C. gariepinus), tilapia (O. niloticus), and common carp (Cyprinus carpio) increased   body weight and length in three fish species (11).
The application   of the peptide during the early stages of fish development should be very important,   The peptide designed by modeling bioinformatics from human ghrelin receptor   and obtained by chemical synthesis is recognized by the secretagogue receptor   GHS-R1a, stimulating the secretion of growth hormone due to the action of growth   hormone results in further development of cells and tissues of the larvae. It   promotes accelerated growth with increased weight concurrently is recognized   by immune system cells and stimulates the innate immune response, the main defense   mechanism of fish during the early stages of life as a result you can get higher   quality larvae
To summarize,   the action of this GHS has been assessed employing in vitro and in   vivo methods, we have demonstrated the effect of a new growth hormone   secretagogue , A228, as a growth hormone secretagogue, that elicits GH in tilapia   pituitary cell culture and also has stimulatory effect on the superoxide anion   production on tilapia PBL and a cell line J774 from mice macrophages .To administrate   the molecule using immersion baths, the weight gain is enhanced in tilapia larvae   . These results support the interrelation between endocrine and immune system.    
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Recibido: 14 de   marzo de 2016.
Aprobado:   20 de mayo de 2016.
Rebeca Martínez1, Katerina Gonzalez2, Alain Gonzalez1, Kenia Ubieta1, Fidel Herrera1, Osvaldo Reyes3, Hilda Garay 3, Ayme Oliva 1, Elsa Rodriguez1, Mario Pablo Estrada1
1 Biotechnology Animal Division, Aquatic Biotechnology Department, CIGB, PO Box 6162, Havana 10600, Cuba.
2 Escuela Cs. Ambientales, Laboratorio Fisiología de Peces. Universidad Católica de Temuco. Chile.
3 Physico-Chemistry Division, Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana 10600, Cuba.
Correspondence : Rebeca Martinez. Email: [email protected]
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