Marked Cortisol Production by Intracrine ACTH in GIP-Treated Cultured Adrenal Cells in Which the GIP Receptor Was Exogenously Introduced
Hiroko FujiiMimi Tamamori‐AdachiKousuke UchidaTakao SusaTakashi NakakuraHaruo HagiwaraMasayoshi IizukaHiroko OkinagaYuji TanakaTomoki Okazaki
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Abstract:
The ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) in the human adrenal gland causes significant hypercortisolemia after ingestion of each meal and leads to Cushing's syndrome, implying that human GIPR activation is capable of robustly activating adrenal glucocorticoid secretion. In this study, we transiently transfected the human GIPR expression vector into cultured human adrenocortical carcinoma cells (H295R) and treated them with GIP to examine the direct link between GIPR activation and steroidogenesis. Using quantitative RT-PCR assay, we examined gene expression of steroidogenic related proteins, and carried out immunofluorescence analysis to prove that forced GIPR overexpression directly promotes production of steroidogenic enzymes CYP17A1 and CYP21A2 at the single cell level. Immunofluorescence showed that the transfection efficiency of the GIPR gene in H295R cells was approximately 5%, and GIP stimulation enhanced CYP21A2 and CYP17A1 expression in GIPR-introduced H295R cells (H295R-GIPR). Interestingly, these steroidogenic enzymes were also expressed in the GIPR (-) cells adjacent to the GIPR (+) cells. The mRNA levels of a cholesterol transport protein required for all steroidogenesis, StAR, and steroidogenic enzymes, HSD3β2, CYP11A1, CYP21A2, and CYP17A1 increased 1.2-2.1-fold in GIP-stimulated H295R-GIPR cells. These changes were reflected in the culture medium in which 1.5-fold increase in the cortisol concentration was confirmed. Furthermore, the levels of adenocorticotropic hormone (ACTH) receptor and ACTH precursor proopiomelanocortin (POMC) mRNA were upregulated 2- and 1.5-fold, respectively. Immunofluorescence showed that ACTH expression was detected in GIP-stimulated H295R-GIPR cells. An ACTH-receptor antagonist significantly inhibited steroidogenic gene expression and cortisol production. Immunostaining for both CYP17A1 and CYP21A2 was attenuated in cells treated with ACTH receptor antagonists as well as with POMC siRNA. These results demonstrated that GIPR activation promoted production and release of ACTH, and that steroidogenesis is activated by endogenously secreted ACTH following GIP administration, at least in part, in H295R cells.Keywords:
CYP17A1
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The physiologic role of adrenocorticotropic hormone (ACTH) and its part in the pituitary—adrenal axis is one of the most intensively studied systems in endocrinology. ACTH was one of the first hormones that was found to stimulate cAMP production by the adrenal gland (1), and the notion that this effect was mediated via a specific cell surface receptor was confirmed by the elegant studies of Lefkowitz and colleagues (2) in work that set a standard for receptor characterization. Nevertheless, progress on the understanding of the ACTH receptor has been relatively slow. It is now clear that the MC2-R is synonymous with the ACTH receptor, and both terms are used in this chapter. In general, the term ACTH receptor is used to describe the functional entity for example, ligand binding to adrenal cells, whereas the term MC2-R is used to describe aspects that can clearly be related to this gene.
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ACTHのradioimmunoassayの際に, 非標識ACTHの増加に伴ない標識ACTHの抗体との結合も増加するという現象が観察された.この現象 (paradoxical binding Phenomenon) の発現機序について考察を加えた.その結果, 特殊な抗血清では, 抗原抗体反応においても酵素基質反応においてみられるアロステリック効果に類似した現象が認められる可能性が示唆された.この現象を利用するとACTHのradidmmunoassayがきわめて鋭敏に可能であつた.
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Crude membranes (20,000 x g pellet) prepared from human, rat, and ovine adrenals bind 1251-corticotropin-(l-24)tetracosapeptide (rz51-ACTH1-& and degrade unbound hormone.The degradation is dependent on temperature and the concentration of membrane proteins.The degradation of 1251-[9-tryptophan(o-nitrophenylsulfenyl)] -corticotropin -(1 -24) -tetracosapeptide (rz51 -NPS -ACTHI-21) is similar to 1251-ACTH1-24, but that of 1251-corticotropin-(11-24)-tetradecapeptide (1251-ACTH11-24) is larger.The degradation of 1251-ACTH1-24 is inhibited by ACTH1-24 and corticotropin-( 1 -IO)-decapeptide (ACTH&, but ACTHll-z4 at the same molar concentration has no effect.On the other hand, the degradation of 1251-ACTH11W2, is protected by ACTHll-24 and ACTHI-24, but not by ACTHlmlO.This suggests two systems of degradation, one will have the NH2-terminal sequence of ACTH,-2, as substrate, and the other the 11-24 COOH-terminal sequence.The main labeled product from the degradation of the 1251-ACTH1-24 and 1251-ACTH11-2, behaves as [1251]monoiodotyrosine on Sephadex G-50 and paper chromatography.The independence of ACTH binding to its receptor and degradation is demonstrated by the following facts.(a) Calcium and pancreatic trypsin inhibitor completely inhibit the binding at concentrations when the degradation is not altered; (b) the sequences of peptides of ACTH which inhibit the binding and degradation of 1251-ACTH,.24 are different.
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The adrenocorticotropic hormone ACTH is a pituitary hormone derived from a larger peptide, the proopiomelanocortine (POMC), as are the MSHs (α-MSH, β-MSH and γ-MSH) and the β-LPH-related polypeptides (Figure 1A). ACTH drives adrenal steroidogenesis and growth of the adrenal gland. ACTH is a 39 amino acids polypeptide that binds and activates its cognate receptor (melanocortin receptor 2, MC2R) through the two regions H6F7R8W9 and K15K16R17R18P19. Most POMC-derived polypeptides contain the H6F7R8W9 sequence that is conserved through evolution. This explains the difficulties in developing selective agonists or antagonists to the melanocortin receptors. In this review, we will discuss the clinical aspects of the role of ACTH in physiology and disease, and potential clinical use of selective ACTH antagonists.
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Adrenocorticotropic hormone (ACTH) is a tropic hormone produced by the anterior pituitary. The hypothalamic-pituitary axis controls it. ACTH regulates cortisol and androgen production. Diseases associated with ACTH include Addison disease, Cushing syndrome and Cushing disease.
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Adrenocorticotropic hormone (ACTH ) is a hormone produced by the pituitary gland that stimulates the production of cortisol. Cortisol is a steroid hormone produced by the adrenal glands that is important for regulating glucose, protein, and lipid metabolism; suppressing the immune system's response; and helping to maintain blood pressure. This process of stimulating ACTH production in the pituitary is done via the hypothalamic-pituitary-adrenal axis (HPA ). Laboratory evaluation of ACTH can be assessed by ACTH . For most accurate results, testing should be done between 6 and 10 a.m. ACTH laboratory tests require a special refrigerated centrifuge to immediately separate plasma from cells, then the specimen needs to be immediately frozen. ACTH concentrations vary considerably depending on physiologic conditions and therefore should always be evaluated simultaneously with cortisol. Different assay methods can vary and cannot be used interchangeably. ACTH can also be measured as part of the ACTH stimulation test.
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