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Updated: Apr 22, 2026
Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions
Published on: March 12, 2019
Orexin-A stimulates 3β-hydroxysteroid dehydrogenase expression and cortisol production in H295R human adrenocortical
Xiaocen Chang1, Yuyan Zhao1, Shujing Ju1
1Departments of Endocrinology and Orthopedic Surgery, The First Affiliated Hospital, China Medical University, Shenyang, Liaoning 110001, P.R. China.
Abstract:
Orexin-A is a regulatory peptide involved in the regulation of food intake, sleep-wakefulness, and it has various endocrine and metabolic functions. It orchestrates diverse central and peripheral processes through the stimulation of two G-protein coupled receptors, orexin receptor type 1 (OX1 receptor) and orexin receptor type 2 (OX2 receptor). In this study, human adrenocortical cells (NCI-H295R cells) were incubated with various concentrations of orexin-A (10-10 to 10-6 M) in vitro, and the mRNA and protein expression of OX1 receptor was determined in the cells. In addition, NCI-H295R cells treated with 10-6 M orexin-A were then treated with or without OX1 receptor specific antagonist (SB334867), AKT antagonist (PF-04691502), or a combination of both. Subsequently, cell proliferation, the cortisol content in the medium and the mRNA and protein expression expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) were analyzed. The activity of the AKT signaling pathway was also determined in the NCI-H295R cells. We observed that the increase in the mRNA and protein expression of OX1 receptor was orexin-A concentration-dependent, with 10-6 M orexin-A exerting the most potent effect. Orexin-A enhanced cell proliferation and cortisol production, and increased the mRNA and protein expression of 3β-HSD in the NCI-H295R cells; however, these effects were partly blocked by the OX1 receptor antagonist, the AKT antagonist and the combination of both. Furthermore, orexin-A significantly increased the phosphorylation of AKT, with the levels of total AKT protein remaining unaltered. This effect was blocked in the presence of PF-04691502 (10-6 M), SB334867 (10-6 M) and the combination of both. On the whole, our data demonstrate that the effects of orexin-A on the survival and function of human adrenocortical cells are mediated through the AKT signaling pathway.
Insights
Orexin-A influences human adrenocortical cells by increasing proliferation and cortisol production via the OX1 receptor and AKT pathway. These orexin-A effects on cell survival and function are mediated through AKT signaling.
Area of Science:
- Endocrinology
- Cell Biology
- Molecular Signaling
Background:
- Orexin-A is a peptide regulating appetite, sleep, and metabolic functions.
- It acts through orexin receptor type 1 (OX1 receptor) and orexin receptor type 2 (OX2 receptor).
- The role of orexin-A in adrenocortical cell function is not fully understood.
Purpose of the Study:
- To investigate the expression of OX1 receptor in human adrenocortical cells.
- To determine the effects of orexin-A on adrenocortical cell proliferation, cortisol production, and 3β-HSD expression.
- To elucidate the involvement of the AKT signaling pathway in mediating orexin-A's actions on adrenocortical cells.
Main Methods:
- Human adrenocortical NCI-H295R cells were treated with varying concentrations of orexin-A.
- OX1 receptor mRNA and protein expression were analyzed.
- Cell proliferation, cortisol content, 3β-HSD expression, and AKT pathway activity were assessed following treatment with orexin-A and specific antagonists (SB334867 for OX1 receptor, PF-04691502 for AKT).
Main Results:
- Orexin-A increased OX1 receptor mRNA and protein expression in a concentration-dependent manner.
- Orexin-A enhanced NCI-H295R cell proliferation, cortisol production, and 3β-HSD expression.
- These effects were partially inhibited by OX1 receptor and AKT antagonists.
- Orexin-A activated the AKT signaling pathway, evidenced by increased AKT phosphorylation, an effect blocked by antagonists.
Conclusions:
- Orexin-A upregulates OX1 receptor expression in human adrenocortical cells.
- Orexin-A promotes adrenocortical cell survival and function, including cortisol production.
- The effects of orexin-A on adrenocortical cells are mediated via the OX1 receptor and the AKT signaling pathway.
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