DOI QR코드

DOI QR Code

The Efficacy of Enhanced Growth by Ectopic Expression of Ghrelin and Its Variants Using Injectable Myogenic Vectors

  • Xie, Q.F. (College of Animal Science and Technology, China Agricultural University) ;
  • Wu, C.X. (College of Animal Science and Technology, China Agricultural University) ;
  • Meng, Q.Y. (State Key Laboratory for Agrobiotechnology, China Agricultural University) ;
  • Li, N. (State Key Laboratory for Agrobiotechnology, China Agricultural University)
  • Received : 2003.02.03
  • Accepted : 2003.10.28
  • Published : 2004.01.01

Abstract

Ghrelin is an acylated peptide recently identified as the endogenous ligand for the growth hormone (GH) secretagogues receptor 1a (GHS-R1a) and is involved in a novel system for regulating GH release. To understand the long-term effects of ghrelin, here we constructed six myogenic expression vectors containing the cDNA of swine mature ghrelin (pGEM-wt-sGhln, pGEM-wt-hGhln), ghrelin mutant of $Ser^3$ with $Trp^3$ (pGEM-mt-sGhln, pGEM-mt-hGhln) and truncated ghrelin derivative (pGEM-tmtsGhln, pGEM-tmt-hGhln) encompassing the first 7 residues of ghrelin (including $Ser^3$ substituted with $Trp^3$) and adding a basic amino acid, Lys (K) in the C-terminus. The constructs, pGEM-wt-sGhln, pGEM-mt-sGhln and pGEM-tmt-sGhln were linked with the ghrelin leader sequence, while the pGEM-wt-hGhln, pGEM-mt-hGhln and pGEM-tmt-hGhln were linked with a leader sequence from the human growth hormone releasing hormone (hGHRH). Intramuscular injection of 200 ${\mu}g$ pGEM-wt-sGhln or pGEM-tmt-sGhln augmented growth over 3 weeks in normal rats and peaked at day 21 or 14 post-injection respectively, whose body weight gains were on average approximately 6% or 19% heavier over controls. However, other injectable vectors had no such enhanced growth effects. Our results suggested that the efficacy of the ghrelin leader sequence was more effective than that of hGHRH in our system. Moreover, the results indicated that skeletal muscle might have the ability to posttranslationally modify the in vivo expressed ghrelin. And the most strikingly, the short ghrelin analog seems to mimic the biological effects more efficiently when compared with the full-length ghrelin.

Keywords

References

  1. Bednarek, M. A., S. D. Feighner, S. S. Pong, K. K. McKee, D. L. Hreniuk, M. V. Silva, V. A. Warren, A. D. Howard, L. H. Van Der Ploeg and J. V. Heck. 2000. Structure-function studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a. J. Med. Chem. 43:4370-4376.
  2. Cassoni, P., M. Papotti and C. Ghe. 2001. Identification, characterization and biological activity of specific receptors for natural (ghrelin) and synthetic growth hormone secretagogues and analogs in human breast carcinomas and cell lines. J. Clin. Endocrinol. Metab. 86:1169-1174.
  3. Cummings, D. E., D. S. Weigle, R. S. Frayo, P. A. Breen, E. P. Dellinger and J. Q. Purnell. 2002. Plasma ghrelin levels after diet-induced weight loss or gastric bypass gastric surgery. N. Engl. J. Med. 346:1623-1630.
  4. Date, Y., M. Nakazato, N. Murakami, M. Kojima, K. Kangawa and S. Matsukura. 2001. Ghrelin acts in the central nervous system to stimulate gastric acid secretion. Biochem. Biophys. Res. Commun. 280:904-907.
  5. Date, Y., N. Murakami, M. Kojima, T. Kuroiwa, S. Matsukura, K. Kangawa and M. Nakazato. 2000. Central effects of a novel acylated peptide, ghrelin, on growth hormone release in rats. Biochem. Biophys. Res. Commun. 275:477-480.
  6. Draghia-Akli, R., M. Fiorotto, L. A. Hill, P. B. Malone, D. R. Deaver and R. J. Schwartz. 1999. Myogenic expression of an injectable protease-resistant growth hormone-releasing hormone augments long-term growth in pigs. Nat. Biotechnol. 17:1179-1183.
  7. Draghia-Akli, R., X. G. Li and R. J. Schwartz. 1997. Enhanced growth by ectopic expression of growth hormone releasing hormone using an injectable myogenic vector. Nat. Biotechnol.15:1285-1289.
  8. Horvath, T. L., S. Diano, P. Sotonyi, M. Heiman and M. Tsch$\"{o}$p.2001. Minireview: ghrelin and the regulation of energy balance-a hypothalamic perspective. Endocrinology. 142:4163-4169.
  9. Kojima, M., H. Hosoda, Y. Date, M. Nakazato, H. Matsuo and K. Kangawa. 1999. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature. 402:656-660.
  10. Lawrence, C. B., A. C. Snape, F. M. Baudoin and S. M. Luckman. 2002. Acute central ghrelin and GH secretagogues induce feeding and activate brain appetite centers. Endocrinology.143:155-162.
  11. Li, S., F. C. Maclaughlin, J. G. Fewell, Y. Li, V. Metha, J. L. Nordstrom, M. Coleman, N. S. Belagali, R. J. Schwartz and L. C. Smith. 1999. Increased level and duration of expression in muscle by co-expression of a transactivator using plasmid systems. Gene Therapy. 6:2005-2011.
  12. Li, X., E. M. Eastman, R. J. Schwartz and R. Draghia-Akli. 1999. Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences. Nat. Biotchnol. 17:241-245.
  13. Masuda, Y., T. Tanaka, N. Inomata, N. Ohnuma, S. Tanaka, Z. Itoh, H. Hosoda, M. Kojima and K. Kangawa. 2000. Ghrelin stimulates gastric acid secretion and motility in rats. Biochem. Biophys. Res. Commun. 276:905-908.
  14. Matsumoto, M., H. Hosoda, Y. Kitajima, N. Morozumi, Y.Minamitake, S. Tanaka, H. Matsuo, M. Kojima, Y. Hayashi and K. Kangawa. 2001. Structure-activity relationship of ghrelin: pharmacological study of ghrelin peptides. Biochem. Biophys. Res. Commun. 287:142-146.
  15. Nagaya, N., K. Miyatake, M. Uematsu, H. Oya, W. Shimizu, H. Hosoda, M. Kojima, N. Nakanishi, H. Mori and K. Kangawa. 2001a. Hemodynamic, renal and hormonal effects of ghrelin infusion in patients with chronic heart failure. J. Clin. Endocrinol. Metab. 86:5854-5859.
  16. Nagaya, N., M. Uematsu, M. Kojima, Y. Ikeda, F. Yoshihara, W. Shimizu, H. Hosoda, Y. Hirota, H. Ishida, H. Mori and K. Kangawa. 2001b. Chronic administration of ghrelin improves left ventricular dysfunction and attenuates development of cardiac cachexia in rats with heart failure. Circulation. 104:1430-1435.
  17. Nakazato, M., N. Murakami, Y. Date, M. Kojima, H. Matsuo, K. Kangawa and S. Matsukura. 2001. A role for ghrelin in the central regulation of feeding. Nature. 409:194-198.
  18. Nou, V., K. Tomoshi, H. inoue, N. Matsunaga, H. Kuwayama and H. Hidari. 2003. Effect of exogenous porcine GH administration on GH response to GH-releasing peptide-2 and GH-releasing hormone in swine. Asian-Aust. J. Anim. Sci. 16:1188-1192.
  19. Pombo, M., C. M. Pombo, A. Garcia, E. Caminos, O. Gualillo, C. V. Alvarez, F. F. Casanueva and C. Dieguez. 2001. Hormonal control of growth hormone secretion. Horm. Res. 55 (Suppl.1):11-16.
  20. Raff, T., M. Van der Giet, D. Endemann, T. Wiederholt and M. Paul. 1997. Design and testing of $\beta$-actin primers for RT-PCR that do not co-amplify processed pseudogenes. Bio. Techniques. 23:456-460.
  21. Reecy, J. M., C. A. Bidwell, G. P. Briley and A. L. Grant. 1996. Structure and regulation of the porcine skeletal $\alpha$-actin-encoding gene. Gene. 180:23-28.
  22. Reecy, J. M., C. A. Bidwell, O. M. Andrisani, D. E. Gerrard and A. L. Grant. 1998. Multiple regions of the porcine alpha-skeletal actin gene modulate muscle-specific expression in cell culture and directly injected skeletal muscle. Anim. Biotechnol. 9:101-120.
  23. Takaya, K., H. Ariyasu, N. Kanamoto, H. Iwakura, A. Yoshimoto, M. Harada, K. Mori, Y. Komatsu, T. Usui, A. Shimatsu, Y. Ogawa, K. Hosoda, T. Akamizu, M. Kojima, K. Kangawa and K. Nakao. 2000. Ghrelin strongly stimulates growth hormone release in humans. J. Clin. Endocrinol. Metab. 85:4908-4911.
  24. Tanwattana, P., S. Chanpongsang and N. Chaiyabutr. 2003. Effects of exogenous bovine somatotrophin on mammary function of late lactating crossbred Holstein cows. Asian-Aust. J. Anim.Sci. 16:88-95.
  25. Tolle, V., P. Zizzari, C. Tomasetto, M. C. Rio, J. Epelbaum and M. T. Bluet-Pajot. 2001. In vivo and in vitro effects of ghrelin/motilin-related peptide on growth hormone secretion in the rat. Neuroendocrinology 73:54-61.
  26. Torsello, A., C. Ghe, E. Bresciali, F. Catapano, E. Ghigo, R. Deghenghi, V. Locatelli and G. Muccioli. 2002. Short ghrelin peptides neither displace ghrelin binding in vitro nor stimulate GH release in vivo. Endocrinology.143:1968-1971.
  27. Tsch$\"{o}$p, M., D. L. Smiley and M. L. Heiman. 2000. Ghrelin induces adiposity in rodents. Nature 407:908-913.