Supplementary material for: Effects of prednisolone administration on clock gene expression and indices of circadian rhythms in healthy human males (DOI:10.1210/clinem/dgaf564)

Effects of prednisolone administration on clock gene expression and indices of circadian rhythms in healthy human subjects Supplementary Table of contents Randomiza�on procedure ......................................................................................................................... 1 Supplementary table 1 .............................................................................................................................. 1 Supplementary table 2 .............................................................................................................................. 2 Supplementary figure 1 ............................................................................................................................. 3 Supplementary figure 2 ............................................................................................................................. 4 Supplementary figure 3 ............................................................................................................................. 5 Randomiza�on procedure Trial medica�on was packed by the principal inves�gator in iden�cal botles, which were subsequently given a color code (blue or red) and number (1 to 10) by an independent nurse. The nurse conducted block randomiza�on by drawing from a bag containing 5 lots each of red and blue, ensuring that 5 par�cipants started with prednisolone and 5 started with placebo in a random patern. The study medica�on botles were packed in individual brown paper bags labelled with ID numbers and a 1 or 2 indica�ng the order. The study inves�gator handed out the brown paper bags. Par�cipants were instructed to take ½ a tablet (12.5 mg) upon waking as close to 07:00 as possible without perturbing their natural sleep:wake patern, and ½ a tablet 12 hours a�er, as close to 19:00. The last three doses were ingested under supervision on the study day. Compliance and �ming of intake was assessed by coun�ng remaining tablets and by par�cipants comple�ng a drug diary. The inves�gator and par�cipants remained blinded un�l full comple�on of the trial. Harms were systema�cally evaluated by inves�gator ques�oning. Supplementary table 1: Sequences for primer pairs and probes used. Gene Probe Color Forward primer Reverse primer HPRT 5’-(56-FAM/ACA GAG GGC /ZEN/TAC AAT GTG ATG GCC/ 3lABkFQ/ -3’ ZEN 5’- CGA GAT GTG ATG AAG GAG ATG G-3’ 5’- GTA ATC CAG CAG GTC AGC AA-3’ BMAL1 5’- /5HEX/AAA CAC CTC /ZEN/ATT CTC AGG GCA GCA /3lABkFQ/-3’ HEX 5’- GCC ACC AAT CCA TAC ACA GA -3’ 5’- AGT ATC TTC CCT CGG TCA CA -3’ PER3 5’ /5TEX615/TCA GCA AGA AAG CAG GAG CAA AGC /3lAbRQSp/-3’ TEX 5’- TCA TCA CCC TAC AGC TCC TAT C -3’ 5’- GCT TAT GCT TCC CTT TCC T -3’ NR1D2 (REV-ERB-β) 5’ /5Cy5/ATC GAG TGC /TAO/ACC TGG GAT GAC AAA /3lAbRQSp/ - 3’ Cy5 5’- GGT AAT CCC AAG AAT GGT GAT CT -3’ 5’- CAC AGT AGA ACC ATG CCA CTA A -3’ NPAS2 5’- /5Cy55/TGA CAA CAG ACG GCA GCA TCA TCT /3BHQ-3/ -3’ Cy5.5 5’- GCT GAT GTT GGA GGC ATT AGA -3’ 5’- CCA TGA CAT CCG ACG GTA AAT -3’ Supplementary table 2: Side effects reported in the study Side effect Placebo n / total n Prednisolone n / total n Any side effect 3/10 5/10 Sleep disturbance 2/10 3/10 Unrest 1/10 2/10 Supplementary figure 1: CONSORT diagram Supplementary figure 2: Graph of interstitial glucose levels in the study period showing more pronounced nocturnal increases in glucose during prednisolone (blue line), peaking around 2-3 AM despite no food intake. Blue and red shadings indicate SE. Dark grey shadings signify nocturnal periods (00:00 to 06:30). Supplementary figure 3: Cortisol and bioactive GC activity during placebo treatment. A) serum levels of total cortisol and bioactive GC activity expressed in cortisol equivalent nmol/l concentrations. B) Ratio between total cortisol and bioactive GC activity expressed in percent with a fitted sine wave curve showing a mean free cortisol (%) of 5.1 (4.8 to 5.4) and an amplitude of 1.2 (0.5 to 1.9) and a fit (r2) of 0.68. 0 10 20 30 40 Serum bioactive GC activity, nmol/l 0 100 200 300 400 Serum total cortisol, nmol/l 23 01 03 05 07 09 11 13 15 16 18 20 22 00 02 04 06 08 22 10 Clock time PLAC PLAC Free