Background: In humans, loss-of-function mutations in Nicotinamide nucleotide transhydrogenase (NNT) cause familial glucocorticoid deficiency, a potentially fatal, adrenal-specific disorder characterized by increased ACTH and reduced cortisol levels. NNT is a highly conserved inner mitochondrial membrane protein, which supplies high concentrations of NADPH for detoxification of reactive oxygen species (ROS) by glutathione and thioredoxin pathways.
Objective and hypotheses: To determine how loss of NNT results in a steroidogenic phenotype and to identify pathways affected upon limited NADPH supply in adrenal cells.
Method: Adrenal RNA was extracted from Nnt-WT, Nnt-KO, and Nnt-BAC (rescue) mice. RNA-sequencing (RNA-Seq) was performed as paired end reads on the Illumina HiSeq2000 platform. Differential expression levels were confirmed by SYBR-Green q-PCR and western blotting.
Results: RNA-Seq analysis revealed no alterations in antioxidant genes of glutathione and thioredoxin pathways (Prdx3, Gpx1, Sod2, Txnrd2, and Gr) but that NNT loss affects expression of key mitochondrial steroidogenic enzymes (CYP11A1 and CYP11B1) with a 25% reduction in mRNA levels and a more pronounced decrease in protein expression. Comparison of RNA-Seq data from Nnt-WT and -MUT mice revealed differential expression (fold change ≥1.5; p value <0.001) of 91 genes that was reversed in the Nnt-BAC suggesting these genes are directly affected by Nnt loss. The 91 genes fell into 12 biological processes by gene ontology analysis with significant enrichment (4.13-fold; P<0.05) of genes involved in stress response including the heat shock proteins Dnajb1, Hsph1, Hspa1a, and Hspa1b. Interestingly α- and β-haemoglobins (Hba-a1, Hba-a2, Hbb-b1, and Hbb-b2) were highly upregulated in the knockout mouse, suggesting a compensatory mechanism to combat oxidative stress, with levels returning to normal in Nnt-BAC rescue mouse.
Conclusion: Our data suggest loss of Nnt and the resultant reduction in NADPH production affects a number of gene networks within the adrenal. It reveals up- or down-regulation of important pathways presumably to combat the sustained adversity due to mitochondrial NADPH restriction and the concomitant increase in ROS that this causes.
01 - 03 Oct 2015
European Society for Paediatric Endocrinology