Expression of corticosteroid hormone receptors, prereceptors, and molecular chaperones in hypothalamic-pituitary-adrenal axis and adipose tissue after the administration of growth promoters in veal calves
Introduction
Since 1988, the use of anabolic steroids and corticosteroids as growth promoters is not permitted in food-producing livestock for safety reasons, in accordance with the European Union (EU) regulations [1]. The range of compounds known to contribute to growth promotion is constantly expanding and illegal practices have moved to the use of low doses or combined mixtures of several compound classes [2].
The EU bans the use of estrogen in animal production, whereas restricts the application of glucocorticoids (GCs), such as dexamethasone (DEX) and prednisolone (PRD), only to therapeutic indication (primary ketosis, disorders of the musculoskeletal system, allergic reactions, skin diseases, and shock). Despite the ban, estrogen and GCs are illegally used as growth promoters at low dosages and for a prolonged period of time. The administration of estrogen significantly improves weight gain (anabolic effect) [3], whereas GCs enhance carcass and meat quality traits, 2 features particularly appreciated by consumers [4]. The corticosteroids, administered through feed, result in improved feed intake, increased live weight gain, and increased water retention and fat content [3]. Because of a synergistic effect when combined with other molecules at low doses, some synthetic corticosteroids (such as DEX) may be used in combination (so-called cocktails) with β-agonists and/or anabolic steroids as illegal growth promoters in cattle [3]. In this context, the anabolic activity of estrogenic compounds has been claimed to be superior to androgens with enhanced growth performance of 5% to 15% in cattle [5]. A relatively high incidence of noncompliance for PRD in bovine husbandry has been reported in the results from National Residue Control Plan performed by EU member states in the 2005 to 2010 period. On the other hand, the use of PRD orally administered alone in illegal schedule is justified by growth-promoting effects in beef cattle during the finishing period, as previously reported [6]. However, owing to nature and potential danger of these residues in food, the EU, to protect the consumer, has been set maximum residue limits [1].
Glucocorticoids are steroid hormones secreted from the adrenal cortex to regulate whole-body metabolic homeostasis, and their production is regulated by the hypothalamic-pituitary-adrenal (HPA) axis with a feedback control system [7]. Glucocorticoids exert specific metabolic influence on the adipose tissue, including the promotion of adipocyte differentiation [14] and induction of lipolysis and adipogenesis [15].
Their biological effects are exerted mainly by binding 2 proximate members of the nuclear receptor superfamily, the glucocorticoid receptor (GR) and the mineralocorticoid receptor, codified by NR3C1 and NR3C2 genes, respectively. Glucocorticoid binding and intracellular trafficking of the receptors are regulated by FKBP prolyl isomerase 4 (FKBP4), an enhancer of the receptor activity, and FKBP prolyl isomerase 5 (FKBP5), a GC-induced negative regulator of the receptors (reviewed in [8]). The FKBP5 gene expression, but not FKBP4, can be induced via GC treatment [[9], [10], [11], [12]], rendering the FKBP5 protein as a component of an ultra-short regulatory loop in steroid signaling.
Local GC levels in target tissues also depend on the expression and function of the prereceptor system: the hydroxysteroid 11-beta dehydrogenases 1 (HSD11B1) regenerates the active form of GCs, whereas the hydroxysteroid 11-beta dehydrogenases 2 (HSD11B2) metabolizes active GCs into inactive derivatives [13].
The aim of this study was to explore the gene expression of the HPA axis and the adipose tissue of veal calves administered PRD or DEX combined with estradiol, attempting to reproduce the situation of illicit treatment in-field. Moreover, the change of the FKBP4 and FKBP5 expression, 2 genes involved in the regulation of corticosteroid receptors activity, was also investigated. The detection of altered cellular signaling is a promising approach to develop new screening methods to identify the misuse of growth promoters, and this study may allow detection of potential biomarkers to implement the National Residue Monitoring Plans of the European countries. In particular, PRD undergoes an extensive biotransformation, making the analytical identification of this drug more difficult. Consequently, new reliable tests would be helpful to reveal PRD abuse in livestock production.
Section snippets
Animals and experimental design
The animals were healthy upon intravitam and postmortem examinations. The trials were authorized through the Italian Ministry of Health [16] and the Ethical Committee of the University of Turin. The carcasses of the treated animals were appropriately destroyed [17].
As previously reported [18], 22 Friesian male veal calves at approximately 4 mo of age were used. The calves were randomly assigned to 3 experimental groups at approximately 5 mo of age. The experimental schedule adopted in this
Results
In the hypothalamus, the DEX administration in combination with estradiol decreased the FKBP5 expression compared with the control group (P < 0.05), whereas no change of the HSD11B1, NR3C1, NR3C2, and FKBP4 expression was observed. No change of all considered genes was detected in PRD-treated group. The HSD11B2 gene expression was not detectable (Fig. 1).
No change of all considered genes was observed in the pituitary gland of calves treated with PRD or DEX in combination with estradiol (Fig. 2).
Discussion
The bioavailability and action of GCs depend not only on circulating levels but also on the tissue-specific intracellular 11β-HSD pathway. In turn, the synthesis of 11β-HSDs is regulated by exogenous GCs. In our study, the administration of DEX in association with estradiol caused an increase in the gene expression of both the HSD11B isoforms in the adrenal glands, whereas only HSD11B1 expression resulted increased in subcutaneous adipose tissue. These data are in disagreement with previous
Acknowledgments
The authors declare no conflicts of interest.
Credit author statement: B.B. and F.T.C. conceived the study. F.T.C. and L.S.C. collected the samples. L.S.C. and S.D. performed the analysis. L.S.C. drafted the manuscript. F.T.C. revised the manuscript.
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