Effects of peptidic growth hormone secretagogue receptor (GHS-R) antagonist [D-Lys3] on some of serum hormonal and biochemical parameters in Wistar rat model

Efeitos do antagonista [D-Lys3] do receptor do peptídeo secretagogo do hormônio do crescimento (GHS-R) sobre alguns parâmetros bioquímicos e hormonais séricos em um modelo em ratos Wistar

Habib Aghdam Shahryar Alireza Lotfi About the authors

Abstracts

Objective

: The present study investigated the effects of different dosages of a GHS-R antagonist [D-Lys3] on some serum hormonal (cortisol, T3 and T4) and biochemical parameters in a rat.

Materials and methods

: Thirty-six 60-day-old male rats were assigned to four treatments. [D-Lys3]-GHRP-6 solutions were infused via intraperitoneal injections. Blood was collected and analyzed.

Results

: The large dosages of a GHS-R antagonist (200 ng/kg BW) caused increases in cortisol, whereas no significant changes occurred when low dosages were injected. There were no significant changes in T3 and T4 following the administration of the GHS-R antagonist, but a considerable increase was observed in blood glucose levels of the groups (G50, G100, and G200 ng/kg BW). There was a significant increase in total protein when the greatest dose was administrated (G200 ng/kg BW). However, total cholesterol, triglycerides, and albumin showed no significant changes.

Conclusions

: Exogenous GHS-R antagonist can cause an increase in glucose and moderate increases in cortisol and total protein, yet it has no significant effect on T3 and T4 levels or on the concentrations of serum lipids. The effect of GHS-R antagonist is not completely adverse to the effects of ghrelin. Further molecular studies are necessary to identify the physiological effects of the peptidic GHS-R antagonist. Arq Bras Endocrinol Metab. 2014;58(3):288-91

Ghrelin receptor; GHS-R; GH-releasing; regulatory peptide; peripheral infusion


Objetivo

: O presente estudo investigou os efeitos de diferentes doses do antagonista do GHS-R [D-Lys3] sobre alguns parâmetros hormonais (cortisol, T3 e T4) e bioquímicos em ratos.

Materiais e métodos

: Trinta e seis ratos machos com 60 dias de idade foram alocados para quatro tratamentos. Soluções de [D-Lys3]-GHRP-6 foram administradas por meio de injeções intraperitoneais e foram coletadas e analisadas amostras.

Resultados

: Doses altas de antagonista de GHS-R (200 ng/kg PC) levaram a aumento do cortisol, enquanto não houve diferença significativa quando foram injetadas doses baixas. Não houve alterações significativas em T3 e T4 depois da administração do antagonista do GHS-R, mas foi observado aumento considerável nos níveis de glicose sanguínea dos grupos (G50, G100 e G200 ng/kg PC). Houve aumento significativo na proteína total quando foi administrada a maior dose (G200 ng/kg PC), entretanto, não foram observadas alterações no colesterol total, nos triglicérides e na albumina.

Conclusões

: O antagonista do GHS-R exógeno pode causar aumento da glicose e aumento moderado do cortisol e proteína total, embora não haja efeitos significativos nos níveis de T3 e T4 ou na concentração de lipídios séricos. O efeito do antagonista de GHS-R não é completamente adverso aos efeitos da grelina. Devem ser feitos outros estudos moleculares para se identificar os efeitos fisiológicos do peptídeo antagonista do GHS-R. Arq Bras Endocrinol Metab. 2014;58(3):288-91

Receptor da grelina; GHS-R; liberação de GH; peptídeo regulador; infusão periférica


INTRODUCTION

The growth hormone secretagogue receptor (GHS-R) is a G protein-coupled receptor that binds with ghrelin. It plays an important role in energy homeostasis, metabolism, and regulation of body weight (1. Smith RG, Leonard R, Bailey AR, Palyha O, Feighner S, Tan C, et al. Growth hormone secretagogue receptor family members and ligands. Endocrine. 2001;14(1):9-14.,2. Petersenn S. Structure and regulation of the growth hormone secretagogue receptor. Minerva Endocrinol. 2003;27(4):243-56.).

Ghrelin receptors can bind with GHS-R antagonists and block them. GHS-R antagonists can be created by manipulating related peptides. For example, a minor change in GHRP-6 (a GH-releasing peptide) by replacing D-lysine with alanine can change the GHS specificity of GHRP-6, and create a new biomolecule with antagonist effects on GHS-R (3. Smith R, Cheng K, Schoen W, Pong S, Hickey G, Jacks T, et al. A nonpeptidyl growth hormone secretagogue. Science. 1993;260:1640-13.). A synthetic peptidic, ghrelin antagonist [D-Lys3] GHRP-6 (His-DTrp-DLys-Trp- DPhe-Lys-NH2) (Figure 1) is widely utilized in vivo and in vitro as the preferred ghrelin receptor (3. Smith R, Cheng K, Schoen W, Pong S, Hickey G, Jacks T, et al. A nonpeptidyl growth hormone secretagogue. Science. 1993;260:1640-13.,4. Patel K, Dixit VD, Lee JH, Kim JW, Schaffer EM, Nguyen D, et al. Identification of ghrelin receptor blocker, D-[Lys3] GHRP-6 as a CXCR4 receptor antagonist. Int J Biol Sci. 2012;8(1):108-17.).

Figure 1
Chemical structure of D-Lys3-GHRP-6.

Ghrelin was identified as the strongest GH-stimulator peptide (5. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature. 402;1999:656-60.). Today, almost all significant endocrine effects of ghrelin as a GH-, PRL- or ACTH-releasing peptide are identified (5. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature. 402;1999:656-60.

. Kaiya H, Kojima M, Hosoda H, Riley LG, Hirano T, Grau EG, et al. Identification of tilapia ghrelin and its effects on growth hormone and prolactin release in the tilapia, Oreochromis mossambicus. Comp Biochem Physiol B Biochem Mol Biol. 2003;135(3):421-9.

. Lotfi A, Shahryar HA, Ghale-Kandi JG, Kaiya H, Ahmadzadeh A. In ovo administration of ghrelin and subsequent prolactin level in newly hatched chicks. J Poult Sci. 2011;48:135-7.
-8. Pecori Giraldi F, Bucciarelli LG, Saccani A, Scacchi M, Pesce S, Losa M, et al. Ghrelin stimulates adrenocorticotrophic hormone (ACTH) secretion by human ACTH-secreting pituitary adenomas in vitro. J Neuroendocrinol. 2007;19(3):208-12.). It was proposed that if ghrelin is an endocrine peptide and has numerous endocrine effects, blocking its receptors may change the endocrine and metabolic profile of the body. To test this theory, different dosages of GHS-R antagonist [D-Lys3] were peripherally injected into laboratory rats.

MATERIALS AND METHODS

This experiment was conducted in the animal room of the Islamic Azad University in 2012. Thirty-six 60-day-old male rats were assigned to 4 treatments and 3 replicates (with 3. Smith R, Cheng K, Schoen W, Pong S, Hickey G, Jacks T, et al. A nonpeptidyl growth hormone secretagogue. Science. 1993;260:1640-13. animals) in each treatment. The experiment was arranged in a completely randomized design (CRD). The study was conducted in compliance with the Animal Welfare Act, and in accordance with ethical rules of Islamic Azad University.

Injection procedure and sampling

[D-Lys3]-GHRP-6 lyophilized powder (Sigma-Aldrich, USA) was dissolved in distilled water according to the manufacturer’s instructions. The prepared solution was infused via intraperitoneal injections. After 48 h, blood samples were taken collected from the heart following anesthesia.

Analysis

Blood samples were centrifuged (1,200 X g, 7 min, 18°C), and serum was prepared with Alcyon 300 auto analyzer (Abbott Park, IL, USA) and its commercial kits (Elisa Pars Azmoon kits, Pars Azmoon Inc., Tehran, for the biochemical assay, and Glory Elisa kits, USA, for the hormonal assay) in order to determine the values of blood biochemical (glucose, total cholesterol, triglycerides, HDL-cholesterol, albumen, total protein) and hormonal parameters (cortisol, T3, and T4). Data from the experiment was evaluated with the SAS software (SAS Inst. Inc., Cary, NC), and the differences between treatments were investigated by Tukey test with P < 0.05 being considered significant.

RESULTS AND DISCUSSION

The administration of great doses of the GHS-R antagonist (200 ng/kg BW) caused cortisol increases in rat serum, whereas no significant changes occurred when low dosages were injected (Table 1). There were no significant changes in T3 and T4 hormones following the administration of the GHS-R antagonist (Table 1).

As seen in table 2, there was a significant increase in blood glucose for groups submitted the administration of the GHS-R antagonist (G50, G100, and G200). A significant increase in total protein was observed when the greatest dose was administrated (G200), whereas total cholesterol, triglycerides, and albumin showed no significant changes (Table 2).

One study showed that the administration of a ghrelin agonist (growth hormone releasing peptide-2) caused transient increases in serum cortisol (9. Laferrère B, Cynthia A, Colleen DR, Cyril YB. Growth hormone releasing peptide-2 (GHRP-2), like ghrelin, increases food intake in healthy men. J Clin Endocrinol Metab. 2005;90(2):611-4.). Wagner and cols. (1010 . Wagner H, Zierden E, Hauss WH. Effects of synthetic somatostatin on endotoxin-induced changes of growth hormone, cortisol and insulin in plasma, blood sugar and blood leukocytes in man. Klin Wochenschr. 1975;53(11):539-41.) showed that the administration of synthetic somatostatin (growth hormone-inhibiting hormone) caused an elevation in serum cortisol levels. In published studies, there was no direct evidence of the stimulatory effect of the GHS-R antagonist on the release of glucocorticoids. The regulatory effect of ghrelin on glucocorticoids is well documented (1111 . Ghigo E. Ghrelin. In chapter 1: Historical milestones. 1st edition. Boston: Kluwer Academic Publishers; 2004. p. 7.,1212 . Martínez-Fuentes AJ, Moreno-Fernández J, Vázquez-Martínez R, Durán-Prado M, de la Riva A, Tena-Sempere M, et al. Ghrelin is produced by and directly activates corticotrope cells from adrenocorticotropin-secreting adenomas. J Clin Endocrinol Metab. 2006;91(6):2225-31.), although the infusion of ghrelin is not able to cause hypercortisolemia (1111 . Ghigo E. Ghrelin. In chapter 1: Historical milestones. 1st edition. Boston: Kluwer Academic Publishers; 2004. p. 7.). In the present study, it seemed that the infusion of greater dosages of the GHS-R antagonist induced a GH-deficient condition, and subsequent increase in cortisol, similar to the study by Wagner and cols. (1010 . Wagner H, Zierden E, Hauss WH. Effects of synthetic somatostatin on endotoxin-induced changes of growth hormone, cortisol and insulin in plasma, blood sugar and blood leukocytes in man. Klin Wochenschr. 1975;53(11):539-41.). On the other hand, ghrelin receptor blockage and the lack of ghrelin effect, as regulatory factors in the release of glucocorticoid, cause a change in cortisol levels (Table 1). Increases in serum glucose in groups submitted to GHS-R antagonist infusion can be the result of cortisol increases (Tables 1 and 2). Chacko and cols. (1313 . Chacko SK, Haymond MW, Sun Y, Marini JC, Sauer PJ, Ma X, et al. Effect of ghrelin on glucose regulation in mice. Am J Physiol Endocrinol Metab. 2012;302(9):E1055-62.) stated that gluconeogenesis and glycogenolysis are increased and insulin sensitivity is improved by the ablation of the ghrelin or growth hormone secretagogue receptor. Ghrelin is a key peptide in the caloric stability of the body and can maintain glucose homeostasis (1414 . Briggs DI, Andrews ZB. A recent update on the role of ghrelin in glucose homeostasis. Curr Diabetes Rev. 2011;7(3):201-7.). In this regard, it was reported that glucose or insulin at physiological concentrations cannot efficiently affect the regulation of ghrelin plasma concentrations. In the present findings, the administration of the GHS-R antagonist caused an increase in serum glucose along with significant increases in total protein (Table 2). Therefore, it is suggested that the body can consume more protein resources for glucose synthesis in ghrelin-deficient conditions induced by the infusion of the GHS-R antagonist. In this regard, Khazali (1515 . Khazali H. Third ventricle ghrelin infusion effect on the metabolic parameters under different energy levels in diets. Iranian J Sci Tech Trans A. 2009;33:31-42.) stated that, in energy deficient conditions, ghrelin can trigger more protein catabolism as energy sources and increase peripheral urea. In conclusion, the peripheral administration of GHS-R can cause an increase in glucose levels and moderate increases in cortisol and total protein rates with no significant effect on T3 and T4 levels or on the concentration of serum lipids. The effect of the GHS-R antagonist is not completely adverse to the ghrelin effect, but it seems that it can moderate the regulatory effect of ghrelin on glucocorticoids and glucose metabolism. The present study investigated numerous serum endocrine and biochemical parameters, but supplementary and comparative molecular studies are necessary for the identification of the physiological effects of the peptidic GHS-R antagonist.

Acknowledgments

: the authors are thankful to Dr. A. Karami Bonary for his assistance in laboratory analyses.

REFERENCES

  • 1
    Smith RG, Leonard R, Bailey AR, Palyha O, Feighner S, Tan C, et al. Growth hormone secretagogue receptor family members and ligands. Endocrine. 2001;14(1):9-14.
  • 2
    Petersenn S. Structure and regulation of the growth hormone secretagogue receptor. Minerva Endocrinol. 2003;27(4):243-56.
  • 3
    Smith R, Cheng K, Schoen W, Pong S, Hickey G, Jacks T, et al. A nonpeptidyl growth hormone secretagogue. Science. 1993;260:1640-13.
  • 4
    Patel K, Dixit VD, Lee JH, Kim JW, Schaffer EM, Nguyen D, et al. Identification of ghrelin receptor blocker, D-[Lys3] GHRP-6 as a CXCR4 receptor antagonist. Int J Biol Sci. 2012;8(1):108-17.
  • 5
    Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature. 402;1999:656-60.
  • 6
    Kaiya H, Kojima M, Hosoda H, Riley LG, Hirano T, Grau EG, et al. Identification of tilapia ghrelin and its effects on growth hormone and prolactin release in the tilapia, Oreochromis mossambicus. Comp Biochem Physiol B Biochem Mol Biol. 2003;135(3):421-9.
  • 7
    Lotfi A, Shahryar HA, Ghale-Kandi JG, Kaiya H, Ahmadzadeh A. In ovo administration of ghrelin and subsequent prolactin level in newly hatched chicks. J Poult Sci. 2011;48:135-7.
  • 8
    Pecori Giraldi F, Bucciarelli LG, Saccani A, Scacchi M, Pesce S, Losa M, et al. Ghrelin stimulates adrenocorticotrophic hormone (ACTH) secretion by human ACTH-secreting pituitary adenomas in vitro. J Neuroendocrinol. 2007;19(3):208-12.
  • 9
    Laferrère B, Cynthia A, Colleen DR, Cyril YB. Growth hormone releasing peptide-2 (GHRP-2), like ghrelin, increases food intake in healthy men. J Clin Endocrinol Metab. 2005;90(2):611-4.
  • 10
    Wagner H, Zierden E, Hauss WH. Effects of synthetic somatostatin on endotoxin-induced changes of growth hormone, cortisol and insulin in plasma, blood sugar and blood leukocytes in man. Klin Wochenschr. 1975;53(11):539-41.
  • 11
    Ghigo E. Ghrelin. In chapter 1: Historical milestones. 1st edition. Boston: Kluwer Academic Publishers; 2004. p. 7.
  • 12
    Martínez-Fuentes AJ, Moreno-Fernández J, Vázquez-Martínez R, Durán-Prado M, de la Riva A, Tena-Sempere M, et al. Ghrelin is produced by and directly activates corticotrope cells from adrenocorticotropin-secreting adenomas. J Clin Endocrinol Metab. 2006;91(6):2225-31.
  • 13
    Chacko SK, Haymond MW, Sun Y, Marini JC, Sauer PJ, Ma X, et al. Effect of ghrelin on glucose regulation in mice. Am J Physiol Endocrinol Metab. 2012;302(9):E1055-62.
  • 14
    Briggs DI, Andrews ZB. A recent update on the role of ghrelin in glucose homeostasis. Curr Diabetes Rev. 2011;7(3):201-7.
  • 15
    Khazali H. Third ventricle ghrelin infusion effect on the metabolic parameters under different energy levels in diets. Iranian J Sci Tech Trans A. 2009;33:31-42.

Publication Dates

  • Publication in this collection
    Apr 2014

History

  • Received
    Sept 2013
  • Accepted
    3 Jan 2014
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