Cortisol and dehydroepiandrosterone (DHEA) are steroids produced by the zona fasciculata (ZF) and reticularis (ZR), respectively, of the adrenal cortex. Both steroids are upregulated in response to adrenocorticotropic hormone (ACTH). Cortisol is a glucorticoid that is important in the regulation of inflammation and metabolism. DHEA is an adrenal androgen important in fetal growth and puberty but tends to decrease gradually after puberty in both men and women. DHEA has various effects on metabolism and immune function including inhibiting the effects of cortisol on some tissues. During the acute phase of stress, cortisol and DHEA rise due to an increase in ACTH released from the anterior pituitary. In contrast, during chronic stress, cortisol remains elevated but DHEA and ACTH levels decrease. Likewise, stress causes serum levels of IL-6 to increase. IL-6 increases cortisol release from the human and bovine adrenal cortex. IL-6 also decreases DHEA release from zona reticularis of the bovine adrenal gland. In humans the effect of IL-6 on DHEA production is still uncertain. To determine a possible mechanism of IL-6 on the zona fasciculata and reticularis, human H294R cells and bovine adrenal tissue were incubated in serum free medium containing IL-6, at various concentrations and incubation intervals. At the end of the incubation interval, mRNA or protein was extracted from the cells or tissue. Standard PCR, real time PCR, and western blot assays were used to determine the effects of IL-6 on the enzymes involved in cortisol and DHEA synthesis, steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein (StAR), and dosage sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1). In human H295R cells and bovine zona fasciculata cells IL-6 caused an increase in SF-1, StAR, P450scc, 17α hydroxylase, 3β-hydroxysteroid dehydrogenase type 2 (3β HSD2), 21 hydroxylase, and 11β hydroxylase mRNA and protein. IL-6 caused DAX-1 mRNA and protein to decrease. These effects were manifest in a time dependent manner. Dose response treatments incubated for 60 min increased SF-1, StAR, P450scc, 17α hydroxylase, 3β HSD2, 21 hydroxylase, and 11β hydroxylase but there was not significant change between the different treatments of IL-6. The bovine zona reticularis stimulated with IL-6 showed a decrease in SF-1, StAR, P450scc, 17α hydroxylase, and 3β HSD2 with an increase in DAX-1 mRNA and protein. This response was manifest in a time dependent manner for both mRNA and protein, and the effect was dose-dependent for mRNA but not protein levels within the 60 min time period. These data provide a mechanism by which increased stress, physical or emotional, which increases IL-6 serum level, could increase cortisol and decrease DHEA. This would account for decreased immune function, increased blood pressure, and changes in metabolism.

Cortisol is a glucocorticoid produced by the zona fasciculata (ZF) of the adrenal cortex. Traditionally, cortisol production and release was seen as being regulated strictly by adrenocorticotropic hormone (ACTH). While this is true of baseline cortisol levels and in response to acute mental stress, the picture is somewhat more complicated in other situations.Interleukin-6 (IL-6) contributes to the maintenance of cortisol levels in situations of prolonged immune or inflammatory stress. AMP activated protein kinase (AMPK) was investigated as a possible mediator of the action of IL-6 or as an independent actor in raising cortisol levels in response to hypoxemic or hypoglycemic stress.5-aminoimidazole-4-carboxamide 1-b-D-ribofuranoside (AICAR) was used to activate AMPK. Bovine ZF tissue fragments were exposed to AICAR alone and together with a known AMPK inhibitor, compound C. Protein or mRNA was then extracted from these tissue fragments. As an indicator of overall steroidogenic activity, these extracts were tested using RT-PCR and western blot assays for relative protein and mRNA levels of steroidogenic acute regulatory (StAR) protein, steroidogenic factor-1 (SF-1), and dosage sensitive sex reversal adrenal hypoplasia congenita gene on the X chromosome, gene 1 (DAX-1). Also a reporter gene assay was performed on H295R cells with a transfected StAR promoter.In bovine ZF tissue fragments, AICAR caused a significant increase of StAR protein and mRNA and SF-1 protein with a decrease of DAX-1 protein in a dose and time dependant manner. DAX-1 mRNA was shown to decrease in response to AICAR administration in a dose dependant manner. AICAR induced increases in StAR protein and SF-1 protein, and the attendant decrease in DAX-1 protein were all shown to be reduced by administration of compound C. This demonstrated that in this situation AICAR is acting through AMPK. When IL-6 was given with compound C the levels of StAR, SF-1, and DAX-1 were significantly reduced from samples treated with IL-6 alone. AICAR exposure also increased StAR promoter activity in a dose and time dependant manner. This AMPK induced increase in steroidogenic activity provides a possible mechanism for increased cortisol during hypoxia and hypoglycemia, and a possible mediator for IL-6 in the ZF.

The adrenal cortex is a crucial endocrine gland in the mammalian stress response. In chronic inflammatory stress, cortisol is elevated whereas adrenal androgens are decreased. Furthermore, ACTH levels have poor correlation with the plasma cortisol in these conditions, thus suggesting that other factors are driving the stress response during chronic inflammatory stress. Interleukin-6 (IL-6), a cytokine which is released during chronic inflammatory stress, is assumed to be one such factor. Thus the biochemical pathways by which IL-6 increases cortisol release from the zona fasciculata (ZF), and decreases adrenal androgen release from the zona reticularis (ZR) were investigated. Since IL-6 activates AMP-activated kinase (AMPK) in skeletal muscle, AMPK was investigated for IL-6- induced effects in ZF and ZR tissue. The effects of AMPK activation and IL-6 exposure on the expression of the steroidogenic proteins, steroidogenic acute regulatory protein (StAR) and cholesterol side chain cleavage enzyme (P450scc), and on the steroidogenic nuclear factors steroidogenic factor-1 (SF-1) and adrenal hypoplasia congenita, critical region on the X chromosome, gene-1 (DAX-1) were investigated. AMPK activation and IL-6 exposure increased the expression of StAR, P450scc, and SF-1, and decreased DAX-1 in the ZF. Meanwhile, AMPK activation and IL-6 exposure decreased the expression of StAR, P450scc, and SF-1, and increased DAX-1 in the ZR. AMPK inhibition blocked the effects of AMPK activation and IL-6 on the ZF and ZR. Activator Protein-1 (AP-1) was the second biochemical intermediate studied since in other tissues AMPK activation increases the expression and phosphorylation of AP-1 subunits. IL-6 stimulation and AMPK activation increased the expression of the AP-1 subunits cFOS, cJUN, JUN B, and JUN D, while increasing the phosphorylation of cJUN in both the ZF and the ZR. These effects were blocked by AMPK inhibition. Inhibition of AP-1 leads to decreased StAR, P450scc, and SF-1, and increased DAX-1 in the ZF. Meanwhile, AP-1 inhibition leads to increased StAR, P450scc, SF-1, and decreased DAX-1 in the ZR. Therefore the AP-1 complex functions as a biochemical intermediate in the IL-6 and AMPK regulation of steroidogenic enzymes in the ZF and ZR. Overall, the results suggest that IL-6 activates AMPK, which increases the expression and phosphorylation of AP-1 subunits in the ZF and the ZR. However, increased AP-1 activation leads to increased StAR and P450scc in the ZF, but decreased StAR and P450scc in the ZR.