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Updated: May 21, 2026

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions
Published on: March 12, 2019
The essential role of the redox balance in adrenal steroidogenesis
Juliana Lourenço Gebenlian1, Aline Faccioli Bodoni2, Fernanda Borchers Coeli-Lacchini2
1Universidade de São Paulo Faculdade de Medicina de Ribeirão Preto Departamento de Pediatria Ribeirão Preto SP Brasil Departamento de Pediatria, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.
Objective:
To analyze the impact of nicotinamide nucleotide transidrogenase (NNT) enzyme impairment on cortisol production, reactive oxygen species (ROS) formation, mitochondrial activity, and mitogen-activated protein kinase 8 (MAPK8) expression in the adrenocortical cell line.
Materials And Methods:
siRNA NNT knockdown was performed in H295R adrenocortical cells to evaluate the effects on cortisol secretion (RIA) and reactive oxygen species (ROS) intracellular production (DCFDA) in basal conditions and after stimulation with 10 uM forskolin. MAPK8 mRNA relative expression and protein localization were evaluated using quantitative PCR (qPCR) and immunofluorescence, respectively.
Results:
NNT knockdown in the H295R adrenal cells specifically reduced NNT RNA and protein levels. Under basal conditions, no changes in ROS intracellular production or cortisol secretion were seen in NNT-depleted H295R cells. However, under forskolin (10 μM), a potent steroidogenesis agent, we observed a marked reduction in cortisol production (p < 0.0001) at the expense of normal ROS intracellular production and increased MAPK8 expression (p = 0.008).
Conclusion:
NNT-deficient adrenocortical cells can maintain mitochondrial homeostasis and cortisol secretion in basal conditions. However, under stress, these cells seem to maintain the redox balance at the expense of impaired steroidogenesis. MAPK8 pathway activation appears to compensate for NNT deficiency.
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