Effect of glucocorticoids pretreatment on steroidogenic capacity of adrenocortical cells isolated from Meishan piglets

The present in vitro experiment was designed to test whether 48 h of pretreatment with glucocorticoids, cortisol, or dexamethasone (DEX), would affect basal and corticotrophin (ACTH) stimulated (24 h) cortisol secretion from primary cultures of pig adrenocortical cells. Cells were divided into six groups: control pretreatment with or without ACTH challenge, cortisol pretreatment with or without ACTH challenge, and DEX pretreatment with or without ACTH. The culture medium and cells were collected at the end of treatment. Cortisol concentration in medium was measured by radioimmunoassay, and protein content of glucocorticoid receptor (GR) and key regulatory factors for steroidogenesis, including melanocortin type 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage cytochrome P450 (P450scc), were detected by Western blot analysis. The results showed that glucocorticoid pretreatment did not affect cortisol secretion under basal condition without ACTH challenge, but significantly enhanced ACTH-stimulated cortisol secretion. Furthermore, the protein content of GR, MC2R, StAR, and P450scc was all increased in groups pretreated with glucocorticoids. These results indicate that adrenocortical cells pretreated with glucocorticoids display higher steroidogenic capacity under ACTH challenge, through the upregulation of GR and other steroidogenic regulatory factors.     

Characterization of adrenal ACTH signaling pathway and steroidogenic enzymes in Erhualian and Pietrain pigs with different plasma cortisol levels

Our previous study demonstrated significant difference in the basal plasma cortisol levels between Erhualian (EHL) and Pietrain (PIE) pigs, implicating fundamental breed difference in adrenocortical function. The objectives of the present study were therefore to characterize the expression pattern of proteins involved in adrenal ACTH signaling and, including melanocortin type 2 receptor (MC2R), cAMP response element binding protein (CREB) and phosphorylated CREB (pCREB), steroidogenic acute regulatory protein (StAR), as well as that of the key enzymes involved in steroidogenesis in EHL and PIE pigs, in association with the plasma corticotrophin (ACTH) and cortisol levels. The plasma concentrations of the substrates for adrenal steroidogenesis, cholesterol and low-density lipoprotein (LDL) cholesterol, did not differ between breeds. Plasma concentration of ACTH and the adrenal contents of MC2R mRNA and protein were similar in two breeds of pigs, whereas the basal plasma concentrations of cortisol in EHL pigs were 1.5 folds higher than that in PIE pigs. The higher basal plasma cortisol levels in EHL pigs were found to be accompanied with the higher expression of ACTH post-receptor signaling components, cAMP, pCREB and StAR, as well as the higher expression of cholesterol side-chain cleavage cytochrome P450 (P450scc), 17alpha-hydroxylase cytochrome P450 (P450(17alpha)), 21-hydroxylase cytochrome P450 (P450c21) and 11beta-hydroxylase cytochrome P450 (P450(11beta)). These results indicated that the enhanced cAMP/PKA/pCREB-signaling system and augmented expression of StAR and steroidogenic enzymes are major attributes to the higher basal plasma cortisol concentrations in pigs.     

Differential actions of metyrapone on the fetal pituitary-adrenal axis in the sheep fetus in late gestation

It is not clear if an increase in intra-adrenal cortisol is required to mediate the actions of adrenocorticotropic hormone (ACTH) on adrenal growth and steroidogenesis during the prepartum stimulation of the fetal pituitary-adrenal axis. We infused metyrapone, a competitive inhibitor of cortisol biosynthesis, into fetal sheep between 125 and 140 days of gestation (term = 147 +/- 3 days) and measured fetal plasma cortisol, 11-desoxycortisol, and ACTH; pituitary pro-opiomelanocortin mRNA and adrenal expression of ACTH receptor (melanocortin type 2 receptor), steroidogenic acute regulatory protein (StAR), 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), cytochrome P450 17-hydroxylase (CYP17), 3beta-hydroxysteroid dehydrogenase, and cytochrome P450 21-hydroxylase mRNA; and StAR protein in the fetal adrenal gland. Plasma ACTH and 11-desoxycortisol concentrations were higher (P < 0.05), whereas plasma cortisol concentrations were not significantly different in metyrapone- compared with vehicle-infused fetuses. The ratio of plasma cortisol to ACTH concentrations was higher (P < 0.0001) between 136 and 140 days than between 120 and 135 days of gestation in both metyrapone- and vehicle-infused fetuses. The combined adrenal weight and adrenocortical thickness were greater (P < 0.001), and cell density was lower (P < 0.01), in the zona fasciculata of adrenals from the metyrapone-infused group. Adrenal StAR mRNA expression was lower (P < 0.05), whereas the levels of mature StAR protein (30 kDa) were higher (P < 0.05), in the metyrapone-infused fetuses. In addition, adrenal mRNA expression of 11betaHSD2, CYP11A1, and CYP17 were higher (P < 0.05) in the metyrapone-infused fetuses. Thus, metyrapone administration may represent a unique model that allows the investigation of dissociation of the relative actions of ACTH and cortisol on fetal adrenal steroidogenesis and growth during late gestation.     

Marked Cortisol Production by Intracrine ACTH in GIP-Treated Cultured Adrenal Cells in Which the GIP Receptor Was Exogenously Introduced

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.     

Ca2+ signal stimulates the expression of steroidogenic acute regulatory protein and steroidogenesis in bovine adrenal fasciculata-reticularis cells

Adrenal glucocorticoid synthesis is stimulated by ACTH or its nitrophenylsulphenyl derivative, NPS-ACTH. Acute stimulation of steroid hormone biosynthesis is highly dependent on the expression of steroidogenic acute regulatory (StAR) protein. To determine the regulatory mechanism of StAR expression in bovine fasciculata/reticularis cells, we analyzed the second messenger systems involved in StAR protein expression using cultured cells activated by ACTH and NPS-ACTH. We concluded that cAMP is not the essential second messenger for StAR protein expression, since NPS-ACTH activated StAR protein expression more than ACTH without increase in cellular cAMP. A 15-lipoxygenase metabolite(s) of arachidonic acid stimulated steroidogenesis without increase in StAR protein expression, since AA-861, a lipoxygenase inhibitor, inhibited steroidogenesis without affecting StAR protein expression. Stimulation of StAR protein expression and the corresponding increase in the steroidogenesis were inhibited by nicardipine in cells treated with ACTH or NPS-ACTH. These data indicate that the dominant second messenger for the stimulation of StAR protein expression is Ca2+. Calmodulin-dependent kinase II inhibitors KN-93 and KN-62 suppressed steroidogenic activity without affecting StAR expression. The protein kinase C inhibitor Ro 31-8220 did not show any effects on StAR expression and steroidogenesis. Calmodulin-dependent kinase II and protein kinase C can therefore be concluded not to be involved in StAR protein expression in bovine cells.     

Infusion of ACTH stimulates expression of adrenal ACTH receptor and steroidogenic acute regulatory protein mRNA in fetal sheep

The late-gestation plasma cortisol surge in the sheep fetus is critical for stimulating organ development and parturition. Increased adrenal responsiveness is one of the key reasons for the surge; however, the underlying mechanisms are not fully understood. Our recent studies suggest that ACTH-mediated increased expression of ACTH receptor (ACTH-R) and steroid acute regulatory protein (StAR) may play a role in enhancing responsiveness. Hence, we examined effects of ACTH infusion in fetal sheep on mRNA expression of these two mediators of adrenal responsiveness and assessed the functional consequences of this treatment in vitro. Fetuses of approximately 118 and 138 days of gestational age (dGA) were infused with ACTH-(1-24) for 24 h. Controls received saline infusion. Arterial blood was sampled throughout the infusion. Adrenals were isolated and analyzed for ACTH-R and StAR mRNA, or cells were cultured for 48 h. Cells were stimulated with ACTH, and medium was collected for cortisol measurement. Fetal plasma ACTH and cortisol concentrations increased over the infusion period in both groups. ACTH-R mRNA levels were significantly higher in ACTH-infused fetuses in both the 118 and 138 dGA groups. StAR mRNA increased significantly in both the 118 and 138 dGA groups. Adrenal cells from ACTH-infused fetuses were significantly more responsive to ACTH stimulation in terms of cortisol secretion than those from saline-infused controls. These findings demonstrate that increases in circulating ACTH levels promote increased expression of ACTH-R and StAR mRNA and are coupled to heightened adrenal responsiveness.     

StAR protein and steroidogenic enzyme expressions in the rat Harderian gland

The Harderian gland (HG) of the rat (Rattus norvegicus) secretes copious amounts of lipids, such as cholesterol. Here we report a study of the expressions of the StAR protein and key steroidogenic enzymes in the HG of male and female rats. The objective of the present investigation was to ascertain (a) whether the rat HG is involved in steroid production starting with cholesterol, and (b) whether the pattern of gene and protein expressions together with the enzymatic activities display sexual dimorphism. The results demonstrate, for the first time, the expression of StAR gene and protein, and Cyp11a1, Hsd3b1, Hsd17b3, Srd5a1, Srd5a2 and Cyp19a1 genes in the rat HG. StAR mRNA and protein expressions were much greater in males than in females. Immunohistochemical analysis demonstrated a non-homogeneous StAR distribution among glandular cells. Hsd17b3 and Cyp19a1 mRNA levels were higher in males than in females, whereas Srd5a1 mRNA levels were higher in females than in males. No significant differences were observed in mRNA levels of Cyp11a1, Hsd3b1 and Srd5a2 between sexes. Furthermore, the in vitro experiments demonstrated a higher 5α-reductase activity in the female as compared to the male HG vice versa a higher P450 aro activity in males as compared to females. These results suggest that the Harderian gland can be classified as a steroidogenic tissue because it synthesizes cholesterol, expresses StAR and steroidogenic enzymes involved in both androgen and estrogen synthesis. The dimorphic expression and activity of the steroidogenic enzymes may suggest sex-specific hormonal effects into the HG physiology.     

Adiponectin (15-36) stimulates steroidogenic acute regulatory (StAR) protein expression and cortisol production in human adrenocortical cells: Role of AMPK and MAPK kinase pathways

Adiponectin is an abundantly circulating adipokine, orchestrating its effects through two 7-transmembrane receptors (AdipoR1 and AdipoR2). Steroidogenesis is regulated by a variety of neuropeptides and adipokines. Earlier studies have reported adipokine mediated steroid production. A key rate-limiting step in steroidogenesis is cholesterol transportation across the mitochondrial membrane by steroidogenic acute regulatory protein (StAR). Several signalling pathways regulate StAR expression. The actions of adiponectin and its role in human adrenocortical steroid biosynthesis are not fully understood. The aim of this study was to investigate the effects of adiponectin on StAR protein expression, steroidogenic genes, and cortisol production and to dissect the signalling cascades involved in the activation of StAR expression. Using qRT-PCR, Western blot analysis and ELISA, we have demonstrated that stimulation of human adrenocortical H295R cells with adiponectin results in increased cortisol secretion. This effect is accompanied by increased expression of key steroidogenic pathway genes including StAR protein expression via ERK1/2 and AMPK-dependent pathways. This has implications for our understanding of adiponectin receptor activation and peripheral steroidogenesis. Finally, our study aims to emphasise the key role of adipokines in the integration of metabolic activity and energy balance partly via the regulation of adrenal steroid production.     

Beta-endorphin, insulin, ACTH and cortisol plasma levels during oral glucose tolerance test in obesity after weight loss

In order to clarify a possible relationship between opioid peptides and glucose homeostasis in obesity we studied Beta-Endorphin (B-Ep), ACTH, cortisol and insulin plasma levels in response to an oral glucose tolerance test (OGTT) in 8 subjects after a hypocaloric diet for 90 days. We obtained through this treatment a weight loss superior to 30% of the initial weight excess (WE) compared with ideal body weight. Moreover, we compared the obtained results with our preliminary study that was performed with the same protocol but without caloric restriction. B-Ep was measured by RIA after silicic acid extraction and G75 Sephadex column chromatography. ACTH, insulin and cortisol were measured directly on plasma by an RIA method. Basal and during OGTT-induced levels of glucose, insulin, ACTH and cortisol decreased in comparison with the values obtained before diet. Conversely, B-Ep remained higher than normal both in the basal condition and during OGTT, and showed values consistently similar to those before diet. These data show that hyperinsulinemia is corrected by weight loss, while hyperbetaendorphinemia remains unchanged. Accordingly, it can be suggested that no direct relationship occurs between hyper-B-Ep-hyper-IRI in obesity. A further insight into the role of hyper-B-Ep in obesity is, thus, necessary, assuming as hypothesis that the increase in B-Ep may be a cause and not a corollary of the polymorphic aspects of obesity.     

Sex differences, developmental changes, response to injury and cAMP regulation of the mRNA levels of steroidogenic acute regulatory protein, cytochrome p450scc, and aromatase in the olivocerebellar system

Compelling evidence has now demonstrated direct biological actions of sex steroids at the cerebellum. Likewise, the expression of key steroidogenic factors, such as the steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc), and aromatase, at this neural site has been reported. Little is known, however, about the regulation of their genes in the cerebellum. Assessment of StAR, P450scc, and aromatase mRNAs in the cerebellum of male and female rats revealed that the expression of these genes is developmentally regulated, with the highest levels at early postnatal ages in both sexes and with significantly higher mRNA levels in postnatal males. Expression of these genes in the female remained unaltered after perinatal androgenization and along the estrous cycle. In contrast, damage of cerebellar afferent neurons of the inferior olivary nucleus evoked a significant increase in StAR, P450scc, and aromatase mRNA levels at this site, as well as a transient elevation in StAR mRNA at the cerebellum. Finally, enhancement of cAMP levels in cultured cerebellar neurons induced a significant increase in StAR and aromatase mRNA levels. In summary, we present herein novel evidence for the developmentally regulated and partially sexually dimorphic pattern of expression of StAR, P450scc, and aromatase genes in the rat cerebellum. These observations, together with the finding that the mRNA levels of these steroidogenic molecules are sensitive to injury and are regulated by intracellular cAMP, strongly suggest that local steroidogenesis is likely to play an important role during development and adaptation to neurodegenerative processes in the olivocerebellar system.     

Salt-inducible kinase-mediated regulation of steroidogenesis at the early stage of ACTH-stimulation

Salt-inducible kinase (SIK), expressed in Y1 mouse adrenocortical tumor cells at an early stage of adrenocorticotropic hormone (ACTH)-stimulation, represses the cAMP-responsive element (CRE)-dependent gene expression of CYP11A and StAR by acting on bZIP domain of CRE-binding protein. ACTH induced the SIK’s nuclear to cytosolic translocation in a PKA-dependent manner. A mutant SIK in which the PKA-dependently phosphorylatable Ser577 had been replaced with Ala could not move out of the nucleus. The degree of CRE-reporter repression by SIK was strong as long as SIK was present in the nucleus. These indicated that intracellular translocation of SIK might be an important factor to determine the time-dependent change in the level of steroidogenic gene expression in ACTH-stimulated cells. Promoter analyses suggested that SIK repressed gene expressions not only of CYP11A and StAR but also of CYP11B1, CYP11B2 and SIK itself. We propose here that SIK is one of important molecule regulating expression of steroidogenic genes in the early phase of ACTH treatment.     

Adrenocortical responses to ACTH in neonatal rats: effect of hypoxia from birth on corticosterone,StAR, and PBR

The adrenocortical response to hypoxia may be a critical component of the adaptation to this common neonatal stress. Little is known about adrenal function in vivo in hypoxic neonates. The purpose of this study was to evaluate adrenocortical responses to ACTH in suckling rat pups exposed to hypoxia from birth to 5-7 days of age compared with normoxic controls. We also evaluated potential cellular controllers of steroidogenic function in situ. In 7-day-old pups at 0800, hypoxia from birth resulted in increased basal (12.2 +/- 1.4 ng/ml; n = 12) and ACTH-stimulated (94.0 +/- 9.4 ng/ml; n = 14) corticosterone levels compared with normoxic controls (basal = 8.3 +/- 0.5 ng/ml; n = 11; stimulated = 51.3 +/- 3.8 ng/ml; n = 8). This augmentation occurred despite no significant difference in plasma ACTH levels in normoxic vs. hypoxic pups before (85 +/- 4 vs. 78 +/- 8 pg/ml) or after (481 +/- 73 vs. 498 +/- 52 pg/ml) porcine ACTH injection (20 microg/kg). This effect was similar in the afternoon at 6 days of age and even greater at 5 days of age at 0800. The aldosterone response to ACTH was not augmented by exposure to hypoxia from birth. Adrenocortical hypoxia-inducible factor (HIF)-1alpha mRNA was undetectable by RT-PCR. Steroidogenic acute regulatory (StAR) protein in adrenal subcapsules (zona fasciculata/reticularis) was augmented by exposure to hypoxia; this effect was greatest at 5 days of age. Peripheral-type benzodiazepine receptor (PBR) protein was also increased at 6 and 7 days of age in pups exposed to hypoxia from birth. We conclude that hypoxia from birth results in an augmentation of the corticosterone but not aldosterone response to ACTH. This effect appears to be mediated at least in part by an increase in controllers of mitochondrial cholesterol transport (StAR and PBR) and to occur independently of measurable changes in endogenous plasma ACTH. The augmentation of the corticosterone response to acute increases in ACTH in hypoxic pups is likely to be an important component of the overall physiological adaptation to hypoxia in the neonate.     

Mechanisms of inhibition of dehydroepiandrosterone upon corticosterone release from rat zona fasciculata-reticularis cells

We have demonstrated that dehydroepiandrosterone (DHEA) acts directly on rat zona fasciculata-reticularis (ZFR) cells to diminish corticosterone secretion by an inhibition of post-cAMP pathway, and decreases functions of steroidogenic enzymes after P450(scc) as well as steroidogenic acute regulatory (StAR) protein expression. However, the mechanisms by which DHEA engages with environmental messenger signals which translate into interfering StAR protein expression are still unclear. This study explored the effects of DHEA on the phosphorylation/activation of extracellular signal-regulated kinases (ERKs). ERK activation resulted in enhancing phosphorylation of steroidogenic factor-1 (SF-1) and increased StAR protein expression. ZFR cells were incubated in the presence or absence of adrenocorticotropin (ACTH), forskolin (FSK), 25-OH-cholesterol, U0126, and H89 at 37 degrees C. The concentration of corticosterone released into the media was measured by radioimmunoassay (RIA). The cells were used to extract protein for Western blot analysis of ERKs or StAR protein expression or immunoprecipitation of SF-1 analysis. The results suggested that (1) ERK pathway of rat ZFR cells might be PKA dependent, (2) ERK activity was required for SF-1 phosphorylation to upregulate steroidogenesis in rat ZFR cells, and (3) DHEA did not affect ERK phosphorylation, however, it attenuated forskolin-stimulated SF-1 phosphorylation to affect StAR protein expression.