The study was a single-center, single-blind, randomized, two-period/crossover clinical trial.Men and women with Addison’s disease for > 12 months on stable cortisol replacement (with HC 15 to 30 mg/day) for ≥ 3 months were eligible for inclusion. Other inclusion criteria were age 20 to 60 years, body mass index 20 to 30 kg/m2, and ability to comply with the protocol procedures. Exclusion criteria were GC replacement therapy for indication other than Addison’s disease, any treatment with sex hormones including contraceptive drugs, treatment with levothyroxine, renal or hepatic failure, significant and symptomatic cardiovascular disease, diabetes mellitus, current infectious disease with fever, and pregnancy or breastfeeding. HC infusion was prepared by adding 0.4 mL of Solu-Cortef® 50 mg/mL to 999.6 mL 0.9% saline, which resulted in 1 mg HC per 50 mL intravenous infusion. HC infusion was adjusted in accordance with previous observations in healthy males and interventions in both sexes. The aim was to achieve a near-physiological circadian cortisol curve with early morning rise in serum cortisol that would peak at 7 AM and trough concentrations at midnight. In the GC-withdrawal intervention, 0.9% saline infusion alone was administered using the same volume as during the HC infusion. Thus, a person weighing 75 kg received 2 L of intravenous infusion over 26 hours during each intervention.
Extracted molecule
total RNA
Extraction protocol
PBMCs were isolated on-site from whole blood using a gradient-based separation procedure and Ficoll-Paque PREMIUM (GE Healthcare). Purified PBMCs were lysed using QIAzol Lysis Buffer (Qiagen, Hilden, Germany) on a QIAshredder column (Qiagen). The lysate was subsequently frozen and stored at –70°C. Samples were eluted in RNAse-free water. RNA concentration was measured spectrophotometrically and the A260/A280 ratio was 1.97 to 2.05.
Label
biotin
Label protocol
Microarray gene expression analysis in both PBMC and adipose tissue was performed at the Array and Analysis Facility, Science for Life Laboratory at Uppsala Biomedical Center (BMC), Sweden. RNA quality was evaluated using the Agilent 2100 Bioanalyzer system (Agilent Technologies Inc., Palo Alto, CA). Total RNA 150 ng from each PBMC sample and 10 ng from each adipose tissue sample were used to generate amplified and biotinylated sense-strand cDNA from the entire expressed genome according to the GeneChip® WT PLUS Reagent Kit User Manual (P/N 703174 Rev. 1, Affymetrix Inc., Santa Clara, CA).
Hybridization protocol
GeneChip® ST Arrays (GeneChip® Human Gene 2.0 ST Array) were hybridized for 16 hours in a 45°C incubator, rotated at 60 rpm. According to the GeneChip® Expression Wash, Stain and Scan Manual (P/N 702731 Rev. 3, Affymetrix Inc.), the arrays were then washed and stained using the GeneChip™ Fluidics Station 450
Scan protocol
Scanned using the GeneChip® Scanner 3000 7G
Description
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Data processing
Gene level summary of the exon array was performed after RMA data processing using quality control of transcriptomic data within Qlucore Omics Explorer 3.3 Principal component analysis (PCA) with cross validation and data consistency was used to confirm data consistency. Differential gene expression was determined by a paired t-test comparing the two interventions. Principal component analysis (PCA) was performed to provide further quality control and to define the relationship of variance between samples, allowing structure within the data set to be defined (Qlucore Omics Explorer 3.3, Lund, Sweden). HuGene-2_0-st.pgf HuGene-2_0-st-v1.na36.hg19.transcript.csv
