A Radioimmunoassay for Rat Ghrelin: Evaluation of Method and Effects of Nonylphenol on Ghrelin Secretion in Force-Fed Young Rats

10.4077/CJP.2011.AMM088

Yen-Jui Chang；Wei-Ju Huang；Han-Wei Lin；Ling-Ling Chang；Full-Young Chang；Paulus S. Wang

anti-ghrelin serum ； rat ghrelin RIA ； serum and tissue ghrelin ； corticosterone ； nonylphenol

The Chinese Journal of Physiology

54卷5期（2011 / 10 / 01）

22 - 29

英文

Antiserum YJC 13-31 against the rat ghrelin conjugated to bovine serum albumin (BSA) was produced in the rabbit and a double antibody radioimmunoassay (RIA) for ghrelin has been developed. Characterization results of this antiserum revealed no cross-reaction with human growth hormone and somatostatin. Weak cross-reactions with insulin (0.1%), rat growth hormone (0.1%) and glucagon (0.3%) were observed, which scarcely interfered the assay system. The sensitivity of this RIA was 5 pg per assay tube. With the rat serum samples, the within-assay precision was 7.1% and the between-assay precision was 12.3%. The RIA was also available to detect the ghrelin in rat tissue extracts with good parallelism to the rat ghrelin standard. In application, the serum ghrelin and corticosterone levels in weaned rats were measured by RIA. Gavage of saline was sufficient to raise serum ghrelin from 2.6±0.18 to 6.7±0.7 ng/ml (P＜0.01). Gavage with nonylphenol (NP) suppressed the elevation of serum ghrelin levels in a dose-dependent manner. Besides, gavages of saline elevated the serum levels of corticosterone from 108.8±13.5 to 188.7±23.5 ng/ml (P＜0.01) but the elevation effects of corticosterone from gavages were overcome by NP in the low dose of 50 mg/kg. It can be speculated that ingestion of NP is harmful to young animals during growth and environmental adaptation.

1. Allred, D.C.,Harvey, J.M.,Berardo, M.,Clark, G.M.(1998).Prognostic and predictive factors in breast cancer by immunohistochemical analysis.Mod. Pathol.,11,155-168.
2. Bassiri, R.M.,Utiger, R.D.(1972).The preparation and specificity of antibody to thyrotropin releasing hormone.Endocrinology,90,722-727.
3. Chang, L.L.,Lo, M.J.,Kan, S.F.,Huang, W.J.,Chen, J.J.,Kau, M.M.,Wang, J.L.,Lin, H.,Tsai, S.C.,Chiao, Y.C.,Yeh, J.Y.,Wun, W.S.,Wang, P.S.(1999).Direct effects of prolactin on corticosterone release by zona fasciculata-reticularis cells from male rats.J. Cell. Biochem.,73,563-572.
4. Chang, L.L.,Wun, W.S.,Wang, P.S.(2010).Effects and mechanisms of nonylphenol on corticosterone release in rat zona fasciculatareticularis cells.Toxicol. Sci.
5. Chang, Y.J.,Chen, M.C.,Chang, F.Y.,Wang, P.S.(2010).Radioimmunoassay for ghrelin: Evaluation of method and the effect of fasting in rats.Adapt. Med.,2
6. Chatterton, R.T., Jr.,Vogelsong, K.M.,Lu, Y.C.,Hudgens, G.A.(1997).Hormonal responses to psychological stress in men preparing for skydiving.J. Clin. Endocrinol. Metab.,82,2503-2509.
7. Cheng, K.C.,Li, Y.X.,Asakawa, A.,Inui, A.(2010).The role of ghrelin in energy homeostasis and its potential clinical relevance (review).Int. J. Mol. Med.,26,771-778.
8. Date, Y.,Kojima, M.,Hosoda, H.,Sawaguchi, A.,Mondal, M.S.,Suganuma, T.,Matsukura, S.,Kangawa, K.,Nakazato, M.(2000).Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans.Endocrinology,141,4255-4261.
9. de la Cour, C.D.,Norlen, P.,Hakanson, R.(2007).Secretion of ghrelin from rat stomach ghrelin cells in response to local microinfusion of candidate messenger compounds: A microdialysis study.Regul. Pept.,143,118-126.
10. Farrace, S.,Biselli, R.,Urbani, L.,Ferlini, C.,De Angelis, C.(1996).Evaluation of stress induced by flight activity by measuring the hormonal response.Biofeedback Self Regul.,21,217-228.
11. Flanagan, D.E.,Evans, M.L.,Monsod, T.P.,Rife, F.,Heptulla, R.A.,Tamborlane, W.V.,Sherwin, R.S.(2003).The influence of insulin on circulating ghrelin.Am. J. Physiol. Endocrinol. Metab.,284,E313-E316.
12. Govoni, N.,De Iasio, R.,Cocco, C.,Parmeggiani, A.,Galeati, G.,Pagotto, U.,Brancia, C.,Spinaci, M.,Tamanini, C.,Pasquali, R.,Ferri, G. L.,Seren, E.(2005).Gastric immunolocalization and plasma profiles of acyl-ghrelin in fasted and fasted-refed prepuberal gilts.J. Endocrinol.,186,505-513.
13. Hosoda, H.,Kangawa, K.(2008).The autonomic nervous system regulates gastric ghrelin secretion in rats.Regul. Pept.,146,12-18.
14. Hosoda, H.,Kojima, M.,Matsuo, H.,Kangawa, K.(2000).Purification and characterization of rat des-gln14-ghrelin, a second endogenous ligand for the growth hormone secretagogue receptor.J. Biol. Chem.,275,21995-22000.
15. Hossaini, A.,Dalgaard, M.,Vinggaard, A.M.,Frandsen, H.,Larsen, J.J.(2001).In utero reproductive study in rats exposed to nonylphenol.Reprod. Toxicol.,15,537-543.
16. Isidori, M.,Lavorgna, M.,Nardelli, A.,Parrella, A.(2006).Toxicity on crustaceans and endocrine disrupting activity on saccharomyces cerevisiae of eight alkylphenols.Chemosphere,64,135-143.
17. Kamegai, J.,Tamura, H.,Shimizu, T.,Ishii, S.,Sugihara, H.,Oikawa, S.(2004).Effects of insulin, leptin, and glucagon on ghrelin secretion from isolated perfused rat stomach.Regul. Pept.,119,77-81.
18. Katayama, T.,Shimamoto, S.,Oda, H.,Nakahara, K.,Kangawa, K.,Murakami, N.(2007).Glucagon receptor expression and glucagon stimulation of ghrelin secretion in rat stomach.Biochem. Biophys. Res. Commun.,357,865-870.
19. Kojima, M.,Hosoda, H.,Date, Y.,Nakazato, M.,Matsuo, H.,Kangawa, K.(1999).Ghrelin is a growth-hormone-releasing acylated peptide from stomach.Nature,402,656-660.
20. Lee, P.C.(1998).Disruption of male reproductive tract development by administration of the xenoestrogen, nonylphenol, to male newborn rats.Endocrine,9,105-111.
21. Li, X.,Ying, G.G.,Su, H.C.,Yang, X.B.,Wang, L.(2010).Simultaneous determination and assessment of 4-nonylphenol, bisphenol a and triclosan in tap water, bottled water and baby bottles.Environ. Int.,36,557-562.
22. Lippl, F.,Kircher, F.,Erdmann, J.,Allescher, H.D.,Schusdziarra, V.(2004).Effect of gip, glp-1, insulin and gastrin on ghrelin release in the isolated rat stomach.Regul. Pept.,119,93-98.
23. Lo, M.J.,Kau, M.M.,Chen, J.J.,Yeh, J.Y.,Lin, H.,Wang, S.W.,Wang, P.S.(1999).Age-related differences in corticosterone secretion in female rats.Metabolism,48,535-541.
24. Lowry, O.H.,Rosebrough, N.J.,Farr, A.L.,Randall, R.J.(1951).Protein measurement with the folin phenol reagent.J. Biol. Chem.,193,265-275.
25. Maestu, J.,Jurimae, J.,Jurimae, T.(2003).Hormonal reactions during heavy training stress and following tapering in highly trained male rowers.Horm. Metab. Res.,35,109-113.
26. Matsumura, N.,Ishibashi, H.,Hirano, M.,Nagao, Y.,Watanabe, N.,Shiratsuchi, H.,Kai, T.,Nishimura, T.,Kashiwagi, A.,Arizono, K.(2005).Effects of nonylphenol and triclosan on production of plasma vitellogenin and testosterone in male south african clawed frogs (xenopus laevis).Biol. Pharm. Bull.,28,1748-1751.
27. McClusky, L.M.,de Jager, C.,Bornman, M.S.(2007).Stage-related increase in the proportion of apoptotic germ cells and altered frequencies of stages in the spermatogenic cycle following gestational, lactational, and direct exposure of male rats to pnonylphenol.Toxicol. Sci.,95,249-256.
28. Mondal, M.S.,Date, Y.,Yamaguchi, H.,Toshinai, K.,Tsuruta, T.,Kangawa, K.,Nakazato, M.(2005).Identification of ghrelin and its receptor in neurons of the rat arcuate nucleus.Regul. Pept.,126,55-59.
29. Musolff, A.,Leschik, S.,Reinstorf, F.,Strauch, G.,Schirmer, M.(2010).Micropollutant loads in the urban water cycle.Environ. Sci. Technol.,44,4877-4883.
30. Nagao, T.,Saito, Y.,Usumi, K.,Nakagomi, M.,Yoshimura, S.,Ono, H.(2000).Disruption of the reproductive system and reproductive performance by administration of nonylphenol to newborn rats.Hum. Exp. Toxicol.,19,284-296.
31. Nagao, T.,Wada, K.,Marumo, H.,Yoshimura, S.,Ono, H.(2001).Reproductive effects of nonylphenol in rats after gavage administration: A two-generation study.Reprod. Toxicol.,15,293-315.
32. Nakai, Y.,Hosoda, H.,Nin, K.,Ooya, C.,Hayashi, H.,Akamizu, T.,Kangawa, K.(2003).Plasma levels of active form of ghrelin during oral glucose tolerance test in patients with anorexia nervosa.Eur. J. Endocrinol,149,R1-R3.
33. Nett, T.M.,Akbar, A.M.,Niswender, G.D.,Hedlund, M.T.,White, W.F.(1973).A radioimmunoassay for gonadotropin-releasing hormone (gn-rh) in serum.J. Clin. Endocrinol. Metab.,36,880-885.
34. Pu, H.F.,Wang, S.G.(1987).Immunoreactive gnrh and trh in the peripheral blood of ovariectomized and ovariectomized-thyroidectomized rats: Relation to lh, tsh and prl release.Chin. J. Physiol.,30,15-27.
35. Roth-Isigkeit, A.,Brechmann, J.,Dibbelt, L.,Sievers, H.H.,Raasch, W.,Schmucker, P.(1998).Persistent endocrine stress response in patients undergoing cardiac surgery.J. Endocrinol. Invest.,21,12-19.
36. Schaller, G.,Schmidt, A.,Pleiner, J.,Woloszczuk, W.,Wolzt, M.,Luger, A.(2003).Plasma ghrelin concentrations are not regulated by glucose or insulin: A double-blind, placebo-controlled crossover clamp study.Diabetes,52,16-20.
37. Stengel, A.,Goebel, M.,Luckey, A.,Yuan, P.Q.,Wang, L.,Tache, Y.(2010).Cold ambient temperature reverses abdominal surgeryinduced delayed gastric emptying and decreased plasma ghrelin levels in rats.Peptides
38. Toshinai, K.,Mondal, M.S.,Nakazato, M.,Date, Y.,Murakami, N.,Kojima, M.,Kangawa, K.,Matsukura, S.(2001).Upregulation of ghrelin expression in the stomach upon fasting, insulin-induced hypoglycemia, and leptin administration.Biochem. Biophys. Res. Commun.,281,1220-1225.
39. Wu, J.J.,Wang, K.L.,Wang, S.W.,Hwang, G.S.,Mao, I.F.,Chen, M.L.,Wang, P.S.(2010).Differential effects of nonylphenol on testosterone secretion in rat leydig cells.Toxicology,268,1-7.

1. Tsai, Su-Ju,Lin, Chuan-Chao,Chang, Chein-Wei(2015).Optimal Effect of Phenol Block in the Sciatic Nerve of Rats: Standardization of Minimized Dosage and Duration of Application.中國生理學雜誌,58(4),237-243.
2. Yu, Ching-Han,Wang, Paulus S.,Wang, Jiing-Rong,Tsai, Shiow-Chwen,Kau, Mei-Mei(2017).Stimulatory Effect of Food Restriction on the Steroidogenesis of Aldosterone in Ovariectomized Rats.中國生理學雜誌,60(2),97-105.