The effects of ACTH on steroid metabolomic profiles in human adrenal cells

Abstract

The adrenal glands are the primary source of mineralocorticoids, glucocorticoids and the so-called adrenal androgens. Under physiological conditions, cortisol and adrenal androgen synthesis are controlled primarily by adrenocorticotrophic hormone (ACTH). Although it is well established that ACTH can stimulate steroidogenesis in the human adrenal gland, the effect of ACTH on overall production of different classes of steroid hormones has not been defined. In this study, we examined the effect of ACTH on the production of twenty-three steroid hormones in adult adrenal (AA) primary cultures and 20 steroids in the adrenal cell line, H295R. Liquid chromatography/tandem mass spectrometry analysis (LC-MS/MS) revealed that in primary cultures that cortisol and corticosterone were the two most abundant steroid hormones produced with or without ACTH treatment (48 h). Cortisol production responded the most to ACTH treatment, with a 64-fold increase. Interestingly, the production of two androgens, androstenedione and 11β-hydroxyandrostenedione, that were also produced in large amounts under basal conditions significantly increased after ACTH incubation. In H295R cells 11-deoxycortisol and androstenedione were the major products under basal conditions. Treatment with forskolin increased the percentage of 11β-hydroxylated products including cortisol and 11β-hydroxyandrostenedione. This study illustrates that normal adrenal cells respond to ACTH through the secretion of a variety of steroid hormones, thus supporting the role of adrenal cells as a source of both corticosteroids and androgens.

H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness

“…LC-MS/MS analysis revealed that the most abundant unconjugated steroid hormones produced under basal conditions by the H295RA cells were 11-deoxycortisol, followed by androstenedione and cortisol. These results are consistent with previous LC-MS/MS steroid profiles reported in H295R cells (Xing et al 2011). The reasons for the relatively mild increase in cortisol after ACTH stimulation in our studies may relate to the low basal expression of CYP11B1 and slow ACTH-induction of the enzyme.…”

“…Steroids produced by the H295R and H295RA cells at baseline and after ACTH stimulation are summarized in Table 2. Similar to our previous study of H295R steroid profiles (Xing et al 2011, Rege et al 2015, the main unconjugated steroids produced in H295RA cells were 11-deoxycortisol, androstenedione, and cortisol, while the mass of aldosterone was low under basal conditions. After ACTH stimulation for 24 h, H295RA cells showed a significant increase in 17a-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, 11-deoxycortisol, pregnenolone sulfate, and 17a-hydroxypregnenolone sulfate as well as aldosterone and cortisol (P!0.05; Table 2).…”

“…The low response to ACTH has been a drawback of this cell model and linked to low level of MC2R in the H295R cells (Mountjoy et al 1994). Clonal selection of H295R cells, specifically with focus on clones with increased ACTH response, has produced HAC15 cells, but these cells do not have the responsiveness seen in primary cultures (Parmar et al 2008, Xing et al 2010, 2011. The existence of an MC2R accessory factor had been suspected because heterologous expression of MC2R does not yield a functional receptor in most cell types (Noon et al 2002).…”

“…The H295R adrenocortical cell line is widely used but is known to have only modest ACTH responsiveness , Rainey et al 2004. Therefore, studies directed toward ACTH action have used primary cultures of adrenal cells (Xing et al 2010(Xing et al , 2011 or Y-1 mouse adrenal cell line, which retains ACTH responsiveness (Schimmer 1979).…”

Prednisolone and prednisone neo-formation in bovine urine after sampling

“…Moreover, adrenocorticotropic hormone regulates adrenal production of cortisol in the zona fasciculata and of androgenic steroids (i.e. dehydroepiandrosterone and ADD) in the zona reticularis (Schimmer and Funder, 2011;Xing et al, 2011), a phenomenon constituting another similarity between the two classes of steroids.…”