Figure 1. Clinical and analytical part of the exploratory study and the replication step.

(A) Subjects with Addison’s disease (primary adrenal insufficiency, step 1) were studied in a random order during both physiological glucocorticoid (GC) exposure and GC withdrawal (step 2). Transcriptomics (whole-genome expression) in peripheral blood mononuclear cells (PBMCs) and adipose tissue (n = 28,869,869 genes), plasma miRNAomics (n = 252), and serum metabolomics in morning samples were analyzed (n = 164) (step 3). Integration of the multi-omic data derived a network including gene expression (derived from two independent tissues), microRNAs (miRNAs), and metabolites that were statistically differentiated between the two interventions (step 4). The miRNA findings, because of their centrality in the network, were replicated in subjects with different GC exposures (within the physiological range) from three independent studies (step 5). (B) Subjects with Addison’s disease (primary adrenal insufficiency) received in a random order intravenous (i.v.) hydrocortisone (HC) infusion mixed in 0.9% saline in a circadian pattern (physiological GC exposure) or the same volume of 0.9% saline alone (GC withdrawal) during 22 hr starting at 9 AM more than 2 weeks apart. During the GC exposure, HC(Solu-Cortef) was administered at a dose of 0.024 mg/kg/hr between 9 AM and 12 PM (first day), 0.012 mg/kg/hr between 12 PM and 8 PM (first day), 0.008 mg/kg/hr between 8 PM and 12 AM (first day), and 0.030 mg/kg/hr between 12 AM and 7 AM (second day). Samples for the ‘omics analyses were collected at 7 AM on day 2 of the intervention (morning samples). p.o.: oral.