The Journal of Clinical Endocrinology & Metabolism, 2023, 108, 1464–1474 https://doi.org/10.1210/clinem/dgac705 Advance access publication 7 December 2022 Clinical Research Article Nicotinamide Nucleotide Transhydrogenase Is Essential for Adrenal Steroidogenesis: Clinical and In Vitro Lessons Aline Faccioli Bodoni, 1, * Fernanda Borchers Coeli-Lacchini, 2, * Juliana Lourenço Gebenlian, 1 Lays Martin Sobral, 3 Cristiana Bernadelli Garcia, 3 Wilson Araújo Silva Jr, 4,5,6 Kamila Chagas Peronni, 4,5,6 Leandra Náira Zambelli Ramalho, 7 Fernando Silva Ramalho, 7 Ayrton C. Moreira, 2 Margaret de Castro, 2 Andreia Machado Leopoldino, 3 and Sonir Roberto Rauber Antonini 1 1 Department of Pediatrics, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP 14049-900, Brazil 2 Department of Internal Medicine, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP 14049-900, Brazil 3 Department of Clinical Analysis, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP 14049-900, Brazil 4 Department of Genetics, Ribeirao Preto Medical School, University of Sao Paulo (USP), Ribeirao Preto, SP 14049-900, Brazil 5 Center for Cell Based Therapy, Ribeirao Preto Medical School, University of Sao Paulo (USP), Ribeirao Preto, SP 14049-900, Brazil 6 Center for Medical Genomics at Clinical Hospital of the Ribeirao Preto Medical School, University of São Paulo, Ribeirao Preto, SP 14049-900, Brazil 7 Department of Pathology and Forensic Medicine, School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP 14049-900, Brazil Correspondence: Sonir R. Antonini, MD, PhD, Ribeirao Preto Medical School, University of Sao Paulo, Avenida Bandeirantes, 3900 Monte Alegre, CEP14049-900 Ribeirao Preto, Sao Paulo, Brazil. Email: antonini@fmrp.usp.br. *These authors have contributed equally to this work. Abstract Context: Nicotinamide nucleotide transhydrogenase (NNT) acts as an antioxidant defense mechanism. NNT mutations cause familial glucocorticoid deficiency (FGD). How impaired oxidative stress disrupts adrenal steroidogenesis remains poorly understood. Objective: To ascertain the role played by NNT in adrenal steroidogenesis. Methods: The genotype–phenotype association of a novel pathogenic NNT variant was evaluated in a boy with FGD. Under basal and oxidative stress (OS) induced conditions, transient cell cultures of the patient’s and controls’ wild-type (WT) mononuclear blood cells were used to evaluate antioxidant mechanisms and mitochondrial parameters (reactive oxygen species [ROS] production, reduced glutathione [GSH], and mitochondrial mass). Using CRISPR/Cas9, a stable NNT gene knockdown model was built in H295R adrenocortical carcinoma cells to determine the role played by NNT in mitochondrial parameters and steroidogenesis. NNT immunohistochemistry was assessed in fetal and postnatal human adrenals. Results: The homozygous NNT p.G866D variant segregated with the FGD phenotype. Under basal and OS conditions, p.G866D homozygous mononuclear blood cells exhibited increased ROS production, and decreased GSH levels and mitochondrial mass than WT NNT cells. In line H295R, NNT knocked down cells presented impaired NNT protein expression, increased ROS production, decreased the mitochondrial mass, as well as the size and the density of cholesterol lipid droplets. NNT knockdown affected steroidogenic enzyme expression, impairing cortisol and aldosterone secretion. In human adrenals, NNT is abundantly expressed in the transition fetal zone and in zona fasciculata. Conclusion: Together, these studies demonstrate the essential role of NNT in adrenal redox homeostasis and steroidogenesis. Key Words: adrenal insufficiency, FGD, NNT, mutations, CRISPR/Cas9 Abbreviations: ATP, adenosine triphosphate; DHEA, dehydroepiandrosterone; FGD, familial glucocorticoid deficiency; FSK, forskolin; GSH, reduced glutathione; GSSG, oxidised glutathione (GSSG); H 2 O 2 , hydrogen peroxide; LH, luteinizing hormone; MC2R, melanocortin-2 receptor; MCM4, mini-chromosome maintenance-deficient 4 homolog gene; MRAP, MC2R accessory protein; NNT, nicotinamide nucleotide transhydrogenase; O − 2 , superoxide anion; OS, oxidative stress; PCR, polymerase chain reaction; ROS, reactive oxygen species; STAR, steroidogenic acute regulatory; TXNRD2, thioredoxin reductase 2; WT, wild type. Received: 11 September 2022. Editorial Decision: 30 November 2022. Corrected and Typeset: 13 January 2023 © The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com Familial glucocorticoid defciency (FGD) is a rare autosomal re- cessive disorder characterized by glucocorticoid and adrenal androgen defciency in the absence of mineralocorticoid def- ciency or with a slight mineralocorticoid impairment. In their frst years of life, patients with FGD usually present with severe hypoglycemia, arterial hypotension, and circulatory shock (1). FGD is caused by mutations affecting the melanocortin-2 receptor (MC2R, OMIM: 607397) or the MC2R accessory protein (MRAP, OMIM: 609196) coding genes (2). Less frequently, FGD results from mutations in the steroidogenic acute regulatory gene (STAR, OMIM: 600617), the mini-chromosome maintenance-defcient 4 homolog gene Downloaded from https://academic.oup.com/jcem/article/108/6/1464/6881692 by guest on 18 May 2023