Glucocorticoids regulate a wide range of biological processes including metabolism. Patients with adrenal insufficiency show impaired glucocorticoid biosynthesis either caused by adrenal defects (primary adrenal insufficiency) or by defects in the pituitary gland or hypothalamus (secondary or tertiary adrenal insufficiency). The systemic consequences of differentially disrupted steroid hormone biosynthesis remain unclear. Increasing evidence suggested steroid hormone precursors can feed into differential pathways and exert biological functions. With similar steroidogenic pathways, zebrafish has been established as an organism for modelling human steroidogenesis. Here, we analyzed the phenotypes of two profound glucocorticoid-deficient zebrafish lines with null mutations in ferredoxin (fdx1b), an electron donor required for mitochondrial steroid biosynthesis, and in 21-hydroxylase (cyp21a2). Both of these glucocorticoid-deficient mutant lines showed low cortisol concentrations, systemic glucocorticoid deficiency as indicated by decreased expression of glucocorticoid-responsive genes (fkbp5, pck1), and enlarged interrenal glands (the counterpart of mammalian adrenal glands). Further in-depth analysis revealed the two glucocorticoid-deficient models showed a distinct oxidative stress response. Antioxidant genes heme oxygenase (hmox1a) and prostaglandin reductase 1 (ptgr1) which are induced by oxidative stress for cell protection are significantly downregulated in cyp21a2−/−, but unchanged in fdx1b−/−, while dual oxidase (duox), involved in formation of superoxide for microbial killing has opposing expression changes in fdx1b−/− and cyp21a2−/−. These observations suggest differential expression is regulated by multiple, distinct pathways rather than directly regulated by glucocorticoids. Interestingly, further induction of oxidative stress by pro-oxidant tert-butylhydroperoxide (tBHQ) induced greater levels of lethal toxicity in wild-type and fdx1b−/− mutants than in cyp21a2−/− mutants, implying oxidative stress may be better buffered in cyp21a2−/− mutants. To elucidate the molecular mechanisms behind this apparent buffering, we performed expression analysis by RT-qPCR on a set of antioxidant genes with tBHQ-treated larvae. hmox1a was upregulated in cyp21a2−/− whilst downregulated in fdx1b−/− and unchanged in wild-type, suggesting hmox1a could be serving as one of the crucial factors in the differential oxidative stress responses. Finally, as a potential route to developing glucocorticoid-deficient models for pharmaceutical screening, we successfully restored the normal expression levels of the majority of antioxidant genes (except for duox) in cyp21a2−/− mutants with both dexamethasone and hydrocortisone. Taken together, our results demonstrate for the first time alterations of oxidative stress and antioxidant balance in glucocorticoid deficiency. Moreover, our results reveal glucocorticoid deficiency due to two different steroidogenic defects has distinct systemic consequences. Thus, our research will contribute to better understand the specific pathophysiological consequences of inborn errors of steroidogenesis.
27 - 29 Sep 2018
European Society for Paediatric Endocrinology