title: Marked cortisol production by intracrine ACTH in GIP-treated cultured adrenal cells in which the GIP receptor was exogenously introduced.
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.
authors.affiliation: Department of General Medicine, National Defense Medical College, Tokorozawa City, Saitama, Japan; Department of Biochemistry, Teikyo University School of Medicine, Tokyo, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Fujii
authors.affiliation: Department of Biochemistry, Teikyo University School of Medicine, Tokyo, Japan.
authors.forename: Mimi
authors.initials: M
authors.lastname: Tamamori-Adachi
authors.affiliation: Department of General Medicine, National Defense Medical College, Tokorozawa City, Saitama, Japan; Department of Biochemistry, Teikyo University School of Medicine, Tokyo, Japan.
authors.forename: Kousuke
authors.initials: K
authors.lastname: Uchida
authors.affiliation: Department of Biochemistry, Teikyo University School of Medicine, Tokyo, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Susa
authors.affiliation: Department of Anatomy, Teikyo University School of Medicine, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakakura
authors.affiliation: Department of Anatomy, Teikyo University School of Medicine, Tokyo, Japan.
authors.forename: Haruo
authors.initials: H
authors.lastname: Hagiwara
authors.affiliation: Department of Biochemistry, Teikyo University School of Medicine, Tokyo, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Iizuka
authors.affiliation: Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Okinaga
authors.affiliation: Department of General Medicine, National Defense Medical College, Tokorozawa City, Saitama, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Department of Biochemistry, Teikyo University School of Medicine, Tokyo, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Okazaki
mesh_terms: D000311:Adrenal Glands / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000324:Adrenocorticotropic Hormone / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D005749:Gastric Inhibitory Polypeptide / Q000494:pharmacology*; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D011964:Receptors, Gastrointestinal Hormone / Q000235:genetics / Q000378:metabolism*; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism; D013255:Steroid 21-Hydroxylase / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011964:Receptors, Gastrointestinal Hormone; D005576:Colforsin; C016276:8-bromocyclic GMP; D005749:Gastric Inhibitory Polypeptide; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; C041086:gastric inhibitory polypeptide receptor; C531510:CYP21A2 protein, human; D013255:Steroid 21-Hydroxylase; C501892:CYP17A1 protein, human; D013254:Steroid 17-alpha-Hydroxylase; D006152:Cyclic GMP; D006854:Hydrocortisone