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.