Growth Hormone-Releasing Peptide-2 (GHRP-2) Acts Synergistically with Growth Hormone-Releasing Hormone (GHRH) to Release Growth Hormone (GH) in Swine

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1 Growth Hormone-Releasing Peptide-2 (GHRP-2) Acts Synergistically with Growth Hormone-Releasing Hormone (GHRH) to Release Growth Hormone (GH) in Swine Long Thang PHUNG1, Nobuyoshi MATSUNAGA, Satoshi HIDAKA, Hideto KUWAYAMA and Hisashi HIDARI Obihiro University of Agriculture and Veterinary Medicine, Obihiro-shi , Japan Hue University of Agriculture and Forestry, 24 Phung 1 Hung, Hue City, Vietnam (Received February 23, 2001; Accepted May 17, 2001) Abstract The aim of the present study was to examine the growth hormone (GH) response to the combined and repeated administrations of growth hormone-releasing peptide-2 (GHRP-2) plus growth hormone releasing hormone (GHRH) in swine. Six cross-bred castrated male swine, days of age were used in this study. Animals were given a single intravenous (i.v.) injection of either GHRP- 5ml saline (served as control) in random order with two days apart. GHRP-2 or GHRH alone and in combination with together stimulated the release of GH in swine. The peak concentrations of GH and areas under the GH response curve (GH AUC) for 120min after these treatments were significantly higher (P<0.01) than those in controls. The combined administration of these two peptides [GHRP- peak concentration of GH and GH AUC for 120min after the second injection were significantly lower (P<0.01) than those after the first injection. These results of the present study indicated that the combined administration of GHRP-2 and GHRH synergistically stimulates the release of GH in swine, and the GH responses to the co-administration of these two peptides decrease after the repeated injection. These findings suggest that GHRP-2 and GHRH act through different receptors and intracellular signal transduction and can be used together for stimulating further release of GH in swine. Animal Science Journal 72 (4): , 2001 Key words: GHRP-2, GHRH, GH, Swine The secretion of growth hormone (GH) is mainly regulated by the functional interplay between two Corresponding: Hisashi HIDARI (fax: +81(0) , hdr@obihiro.ac.jp) Anim. Sci. J. 72 (4): , hypothalamic peptides, one stimulatory, growth hormone-releasing hormone (GHRH) and one inhibitory, somatostatin25, 26) In addition to the studies on the GHRH analogues, a series of synthetic, five to seven amino acid peptides called GH-releasing peptides (GHRPs) were designed23) in the early 1980's, that stimulate the secretion of GH in animals as well as in humans1, 4, 7, 14, 8, 15, 17, 21, Among 24) GHRPs, new hexapeptide GH-releasing peptide-2 (GHRP-2, also named KP102) is the most potent member of the family of GHRPs20, 27). The GH-releasing effect of GHRP-2 is two to three folds more effective in rats

2 PHUNG, MATSUNAGA, HIDAKA, KUWAYAMA and HIDARI and humans than GHRP-6 and GHRP-18). In domestic animals, GHRP-2 has been reported to stimulate the release of GH and improve average daily gain in calves31). Our previous studies also demonstrated that GHRP-2 stimulates the release of porcine GH28, 29) and enhances growth performance28) in swine. There are several in vitro and in vivo evidence for GHRPs acting directly at both the pituitary and hypothalamic levels to release GH2, 3, 6, 9, 10, 14, 15) Moreover, GHRPs and GHRH administered together synergistically stimulate the release of GH5, 6, 11) and their effects are suggested to be mediated via different receptors and intracellular signal transduction pathways3, 11, 16, 32) The synergistic action of GHRP-2 with GHRH to release of GH was reported in rats33) and humans30), but did not in goats18). reason for the discrepancy A possible due to the species difference is still unclear. It is therefore important to clarify the GH response to the combined administration of GHRP-2 and GHRH in swine for effective usage of the peptides in meat production. The present study was designed to determine GH response to the combined and repeated administrations of GHRP-2 and GHRH Peptides in swine. Materials and Methods NH2; lot HQ 008) was supplied by Kaken Pharmaceutical Co. Ltd., Tokyo, Japan. GHRH (human GHRH (1-29) amide; Lot 53H49501) was purchased from Sigma Chemical Co. Louis, MO, USA. Doses response to repeated co-administration of GHRP-2 and GHRH in swine, animals were given two succes- Anim. Sci. J. 72 (4): , were housed in individual pens throughout the experiment and fed a commercial diet containing 16% crude protein ad libitum twice a day at 0900h and 1700h. Water was available at all times. Two days prior to the initiation of treatment, all animals were anesthetized with an intramuscular injection of Ketamin Hydrochloride (10mg/kg BW, Veterinary and an indwelling catheter was inserted into the jugular vein of each animal34). Heparinized saline was flushed daily through the jugular catheter to keep it patent throughout the experiment. Experimental design To investigate GH response to the combined administration of GHRP-2 and GHRH in swine, six crossbred castrated male swine were given a single i.v. trol) at 0900h on the different days of treatment. The experimental animals were not given feed for 16 h before and during the treatment, and had free access to water. Blood samples (2ml each) were collected at -15, 0, 5, 10, 15, 20, 30, 45, 60, 90 and 120min from the injection through indwelling jugular catheters into heparinized syringes and were immediately transferred into centrifuge tubes containing heparin (10IU/ml) and chilled with ice. Blood samples were for GH. In addition, to determine the change in GH Blood samples were collected and treated similarly to those described for single injection of the peptides. All treatments were given in random order and 2 days separated each successive treatment. All experimental protocols and animal care were conducted in accordance with the "Guidelines for the Care and Use of Experimental Animal of Obihiro University of Agriculture and Veterinary Medicine".

3 Radioimmunoassay (RIA) Plasma concentration of pgh was measured at each time point in duplicate using a double-antibody RIA technique28) with pgh antiserum (Lot AFP ) and pgh (Lot AFP-10864B; supplied by Dr. A.F. Parlow, National Hormone and Pituitary Program (NIDDK), Harbor-UCLA Medical Center, Torrance, CA). Porcine GH was used for reference standard and was radioiodinated by the chloramine T method. Briefly, 0.1ml of pgh antiserum (1; 1600,000 final antiserum dilution) diluted in PBS (0.01M; ph 7.5)-EDTA (0.05M) containing 0.2% normal monkey serum (Primate Research Institute, Kyoto University, Japan) was dispensed into assay tubes containing 0.5ml PBS (0.01M; ph 7.5)-BSA (2%) and 0.1ml of sample or standard. Samples were incubated at room temperature for 24h, and 0.1 ml 125I-labeled pgh (approximately 12,000cpm) was added. The incubation was continued for a further 24h, and 0.2ml 2.5% Goat anti-monkey IgG (H&L) (Lot 10TA14Y; Antibodies Incorporated, Davis, California, USA) containing 5% polyethylene glycol 6000 was added. Samples were incubated at room temperature for 24h, and centrifuged at 3,000rpm Porcine GH Responses to GHRP-2 Plus GHRH decanted and pellets were counted using a gamma counter. The sensitivity of the assay was 0.16ng/ml and intra-assay coefficient of variation was 9.9%. Statistical analysis under GH response curve (GH AUC) for 120min after the administration of either GHRP-2, GHRH, and GHRP-2 plus GHRH or saline, as well as after the repeated co-administration of GHRP-2 and GHRH were calculated using the trapezoidal method. Peak concentrations of GH were the highest concentrations attained after treatments. Paired Student's t-test was used to access the significant differences in mean peak concentrations of GH and GH AUC between single injections of the peptides or between the first and second injections of GHRP-2 plus GHRH in repeated administration. Values of P<0.05 were considered statistically significant. Anim. Sci. J. 72 (4): , Results GH responses to a single i.v. injection of GHRP-2, GHRH, and GHRP-2 plus GHRH GH responses following a single i.v. injection of swine is shown in Fig. 1 and Table 1. The injection of saline did not alter the basal plasma concentration of GH, however, injection of GHRP-2, GHRH and GHRP-2 plus GHRH significantly stimulated GH release. After the i.v. injection of these two peptides alone or in combination with together, plasma concentrations of GH rose above basal, peaked at 15min and gradually declined to basal concentrations at 60min for GHRH and within min for GHRP-2 and GHRP-2 plus GHRH (Fig. 1). Compared to the saline control group, the administration of GHRP-2, GHRH and GHRP-2 plus GHRH significantly (P< 0.01) increased peak concentrations of GH and GH AUC for 120min after the treatments (Table 1). To evaluate the possible synergistic effect between GHRP-2 and GHRH, GH AUC for 120min after the combined administration of GHRP-2 and GHRH was

4 PHUNG, MATSUNAGA, HIDAKA, KUWAYAMA and HIDARI Table 1. Effect of single injection of either GHRP-2, GHRH and GHRP-2 plus GHRH or saline on peak concentration of GH and GH response curve (AUC) for 120min after injection in swinea compared to the arithmetic sum of GH AUC after treatment with GHRP-2 and GHRH alone. GH AUC for 120min after the combined administration of these two peptides was significantly (P<0.05) larger than the arithmetic sum of GH AUC following administration of GHRP-2 and GHRH alone, suggesting that the effects between the two peptides on GH secretion was synergistic in swine. GH responses to repeated i.v. injections of GHRP-2 plus GHRH GH responses to two successive i.v. injections of at 2h interval to cross-bred castrated male swine are shown in Fig. 2 and Table 2. The repeated injection of GHRP-2 plus GHRH stimulated the release of GH after each injection. The plasma concentrations of GH peaked at 15min and returned to basal concentration at 120min after the injection of GHRP-2 plus GHRH for the first and within 60-90min for the second injection. The mean peak concentrations of GH and GH AUC for 120min after the second injection of GHRP-2 plus GHRH were significantly (P< 0.01) lower than those after the first injection (Table 2). Discussion These results of the present study confirm the potent GH secreting properties of GHRP-2 in swine, and demonstrate that the combined administration of GHRP-2 and GHRH stimulates synergistically GH release in swine. co-administration after the repeat injection. In addition, GH responses to the of GHRP-2 and GHRH attenuate The stimulatory effect of GHRP-2 on GH secretion Anim. Sci. J. 72 (4): , has been documented in various animal species18, 31, 19, 33) as well as in humans8, 27, 30). Recently, we have also reported that GHRP-2 stimulated the release of GH in swine after i.v., subcutaneous28) and even oral administration29). In addition, the synergistic effect for GH release by the combined administration of GHRP-2 and GHRH has been described in rats33) and humans30). The ability to synergize with GHRH makes a major contribution to the GHreleasing effect of GHRP-2 in vivo. To our knowledge, there was no report before on the effect of combined administration of GHRP-2 and GHRH on GH release in swine. In the present study, our data show that a single i.v. injection of GHRP-2, GHRH, and GHRP-2 plus GHRH stimulated GH release in

5 Porcine GH Responses to GHRP-2 Plus GHRH Table 2. Effect of repeated injection of GHRP-2 plus GHRH on peak concentration of GH and GH AUC for 120min after each injection in swinea swine. The GH AUC for 120min after the combined administration of GHRP-2 and GHRH was significantly larger than either of GHRP-2 or GHRH given alone, also the arithmetic sum of both GH AUCs. It seems to show the synergistic effect of GHRP-2 and GHRH to release GH in swine. A number of hypotheses have been proposed for the synergistic effect of GHRPs and GHRH on GH secretion. Bowers et al.6) suggested that GHRPs release an unknown endogenous hypothalamic factor (Ufactor), which interacts with GHRH on the pituitary to release GH synergistically. This factor requires the presence of GHRH in order to exert an effect on the pituitary. This hypothesis is supported by the synergistic effect for GH release in the GHRPs plus GHRH studies in vivo5, 6, 8, 11), and by the absence of synergism in almost all GHRPs plus GHRH studies in vitro6, 8, 32). Although GHRPs do not influence the release of somatostatin13), they may play an important role in inhibiting the pituitary action of somatostatin12, 13). It has been believed that GHRP-2 has its own receptors in pituitary and hypothalamus. Another possible hypothesis for the synergistic effect between GHRPs and GHRH is that GHRPs may stimulate the endogenous GHRH release13) via its receptors in hypothalamus which would cause further GH release from pituitary. The original GHRP-65, 6) or GHRP-18) administered together with GHRH synergistically stimulated the release of GH in rats6) and humans5, 8). These results in rat, human and swine in our experiment are in contrast to the result obtained by Hashizume et al.18), who found no synergistic effect between GHRP-2 and GHRH on GH secretion in goats. This discrepancy between animal species can not fully explain. The mechanism for the synergistic action of GHRPs and GHRH has remained to elucidate. In the present study, the data also shows that the repeated co-administration of GHRP-2 and GHRH to swine at 2h interval stimulated GH release after each injection. Although GH secretory patterns after the second injection of GHRP-2 plus GHRH are similar to those after the first injection, peak concentration of GH and GH AUC were significantly lower after the second injection than after the first injection. These findings indicate that the GH-releasing effects of GHRP-2 plus GHRH in swine decrease after repeated administration. Massoud et al.22) have also reported a similar result of GH response decrease when administered repeatedly hexarelin (a GHRP) with GHRH in healthy adult men. There is a paucity of data on GH response to repeated co-administration of GHRPs and GHRH. It is difficult to explain the mechanism of diminished GH response after the repeated administration of GHRP-2 plus GHRH from our present experiment, but it may be the result of a partial desensitization of somatotrophs to GHRP-2, down-regulation of receptors, depletion or feedback inhibition of the hypothetical hypothalamic factor or failure of GHRP-2 to cause further endogenous GHRH release. The massive GH release following the first injection of GHRP-2 plus GHRH may have partially depleted the pituitary reserves, thus limiting the amount of GH available for further release in response to a second injection of the two peptides. This has also been suggested in another study22). In summary, these results of the present study demonstrated that the combined administration of Anim. Sci. J. 72 (4): ,

6 PHUNG, MATSUNAGA, HIDAKA, KUWAYAMA and HIDARI GHRP-2 and GHRH to swine synergistically stimulates GH release, and GH responses to the co-administration of these two peptides decrease after repeated injection. These findings suggest that GHRP-2 and GHRH act independently, and can be used together for stimulating further release of GH in swine. Further studies are needed to investigate the mechanism of synergistic action between GHRP-2 and GHRH, and long-term effect of GHRP-2 plus GHRH administration on GH release in swine. Acknowledgments The authors would like to thank Dr. A.F. Parlow and the National Hormone and Pituitary Program (NIDDK) for providing reagents and procedures for pgh RIA, Kaken Pharmaceutical Co. Ltd., Tokyo, Japan for providing GHRP-2, and the Primate Research Institute, Kyoto University, Japan for supplying normal monkey serum. References 1) Argente J, Garcia-segura LM, Pozo J, Chowen JA. Growth hormone-releasing peptides: clinical and basic aspects. Neuroendocrinology, 46: ) Badger TM, Millard WJ, McCormick GF, Bowers CY, Martin JB. The effects of growth hormone (GH)-releasing peptides on GH secretion in perifused pituitary cells of adult male rats. Endocrinology, 115: ) Blake AD, Smith RG. Desensitization studies using perfused rat pituitary cells show that growth hormone-releasing hormone and His-D-Trp-Ala- Trp-D-Phe-Lys-NH2 stimulate growth hormone release through distinct receptor sites. Journal of Endocrinology, 129: ) Bowers CY, Momany FA, Reynolds GA, Hong A. On the in vitro and in vivo activity of a new synthetic hexapeptide that acts on the pituitary to specifically release growth hormone. Endocrinology, 114: ) Bowers CY, Reynolds GA, Durham D, Barrera CM, Pezzoli SS, Thorner MO. Growth hormone (GH) -releasing peptide stimulates GH release in normal men and acts synergistically with GH-releasing hormone. Journal of Clinical Endocrinology and Metabolism, 70: ) Bowers CY, Sartor AO, Reynolds GA, Badger TM. On the actions of the growth hormone-releasing hexapeptide, GHRP. Endocrinology, 128: ) Bowers CY, Alster DK, Frentz JM. The growth hormone-releasing activity of a synthetic hexapeptide in normal men and short statured children after oral administration. Journal of Clinical Endocrinology and Metabolism, 74: ) Bowers CY. GH releasing peptides-structure and kinetics. Journal of Prediatric Endocrinology, 6: ) Camanni F, Ghigo E, Arvat E. Growth hormonereleasing peptides and their analogs. Frontiers in Neuroendocrinology, 19: ) Casanueva FF, Dieguez C. Growth hormone secretagogues: Physiological role and clinical utility. Trends in Endocrinology and Metabolism, 10: ) Cheng K, Chan WWS, Barreto A, Convey EM, Smith RG. The synergistic effects of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 on growth hormone (GH)-releasing factor stimulated GH release and intracellular adenosine 3', 5'-monophosphate accumulation in rat primary pituitary cell culture. Endocrinology, 124: ) Clark RG, Carlsson L, Trojinar J, Robinson ICAF. The effect of growth hormone-releasing peptide and growth hormone-releasing factor on conscious and anaesthetized rats. Journal of Neuroendocrinology, 1: ) Guillaume V, Magnan E, Cataldi M, Dutour A, Sauze N, Renard M, Razafindraibe H, Conte-devoix B, Deghenghi R, Lenaerts V, Oliver C. Growth hormone (GH)-releasing hormone secretion is stimulated by a new GH-releasing hexapeptide in sheep. Endocrinology, 135: ) Ghigo E, Arvat E, Rizzi G, Goffi S, Grottoli S, Mucci M, Boghen MF, Camanni F. Growth hormone-releasing activity of growth hormonereleasing peptide-6 is maintained after short-term oral pretreatment with the hexapeptide in normal aging. European Journal of Endocrinology, 131: ) Ghigo E, Arvat E, Muccioli G, Camanni F. Growth hormone-releasing peptides. European Journal of Endocrinology, 136: ) Goth MI, Lyons CE, Canny BJ, Thorner MO. Pituitary adenylate cyclase activating polypeptide, growth hormone (GH)-releasing peptide and GHreleasing hormone stimulate GH release through Anim. Sci. J. 72 (4): ,

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