Participants
Thirty-two healthy white men were recruited via local advertisements. All participants were in good health as confirmed by medical history, physical examination, screening blood tests and a 75 g 2 h OGTT, performed at screening visit. Inclusion criteria included: age 18–35 years; BMI 20–25 kg/m2; and normoglycaemia as defined by fasting plasma glucose (FPG) < 5.6 mmol/l and 2 h glucose < 7.8 mmol/l during OGTT. Exclusion criteria were the presence of any disease, use of any medication, first-degree relative with type 2 diabetes, smoking, shift work, a history of GC use, excessive sport activities (i.e. more often than twice/week) and changes in weight in the 3 months prior to study participation. The study was approved by an independent ethics committee and the study was conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent before participation.
Study design
The study was a randomised double-blind placebo-controlled dose-response intervention study. Following assessment of eligibility and baseline measurements, participants were randomised to receive either prednisolone 7.5 mg once daily (n = 12), prednisolone 30 mg once daily (n = 12), or placebo (n = 8) treatment for a period of 14 days using block randomisation, as carried out by the department of experimental pharmacology of the VU University Medical Centre. On day −2 and day 13 of treatment, body composition, body fat distribution and liver fat content were quantified. On day −1 and on day 14 of treatment, glucose kinetics, lipolysis and proteolysis were measured in the basal state and during a two-step hyperinsulinaemic–euglycaemic clamp using stable isotopes (electronic supplementary material [ESM] Fig. 1a, b). All measurements were conducted following a 12 h overnight fast with the individuals in the semi-supine position. Participants refrained from drinking alcohol for a period of 24 h before the study days and did not perform strenuous exercise for a period of 48 h before the study days. During all visits, including a follow-up visit at day 7 of treatment, safety and tolerability were assessed. A patient flow diagram is shown in ESM Fig. 2.
Assessment of body composition/body fat distribution
Body composition was measured by dual energy X-ray absorptiometry (DEXA) scans (Delphi A; Hologic, Waltham, MA, USA). Magnetic resonance imaging (MRI), for determination of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) area at the level of L3-L4, and proton magnetic resonance spectroscopy (1H-MRS), to quantify liver fat content, were performed using a 1.5 T MRI scanner (Sonata; Siemens, Erlangen, Germany), as described previously by Tushuizen et al. [17, 18]. All magnetic resonance examinations (DVR) and quantification of abdominal fat compartments (MHM) were done by a single experienced investigator.
Clamp
After an overnight fast of 12 h, participants were admitted to the Clinical Research Unit at 07:30 hours. An indwelling cannula was inserted into an antecubital vein for infusion of stable-isotope tracers, glucose and insulin. To obtain arterialised venous blood samples, a retrograde cannula was inserted in a contralateral wrist vein and maintained in a thermoregulated box at 50°C. To keep the sampling line patent 0.9% NaCl was infused. [6,6-2H2]Glucose, [1,1,2,3,3-2H5]glycerol and L-[1-13C]valine were used as tracers (>99% enriched; Cambridge Isotopes, Andover, MA, USA) to study glucose kinetics, lipolysis and valine turnover respectively. At t = 0 h (08:00 hours), blood samples were drawn for determination of background enrichments. Then, a primed continuous infusion of isotopes was started: [6,6-2H2]glucose (prime: 11 μmol/kg; continuous: 0.11 μmol kg−1 min−1), [1,1,2,3,3-2H5]glycerol (prime: 1.6 μmol/kg; continuous: 0.11 μmol kg-1 min-1), and L-[1-13C]valine (prime: 13.7 μmol/kg; continuous: 0.153 μmol kg−1 min−1) and continued until the end of the clamp. After a 2 h equilibrium period (14 h of fasting), three blood samples were drawn for determination of basal glucose concentrations, isotope enrichment, and levels of glucoregulatory hormones and NEFA. Thereafter, a two-step hyperinsulinaemic–euglycaemic clamp was started: step 1 included an infusion of insulin at a rate of 20 mU m−2 min−1 (Actrapid 100 U/ml; Novo Nordisk, Alphen aan den Rijn, the Netherlands) to assess hepatic insulin sensitivity. Glucose 20% was started to maintain a plasma glucose concentration of 5 mmol/l. [6,6-2H2]Glucose was added to the glucose solution to achieve glucose enrichments of 1% in order to approximate the values for enrichment reached in plasma and thereby minimising changes in isotopic enrichment due to changes in the infusion rate of exogenous glucose. Plasma glucose concentrations were measured every 5 min at bedside. After 2 h of insulin infusion, five blood samples were drawn at 5 min intervals for the measurement of glucose concentrations and isotopic enrichments. Another blood sample was drawn for measurement of glucoregulatory hormones and NEFA. Hereafter, insulin infusion was increased to a rate of 60 mU m−2 min−1 (step 2) to assess peripheral insulin sensitivity. After 2 h of insulin infusion, blood sampling for glucose, isotope enrichments, glucoregulatory hormones and NEFA was repeated (ESM Fig. 1b).
Indirect calorimetry
Oxygen consumption (\( {\mathop V\limits^ \cdot }{\text{O}}_{2} \)) and carbon dioxide production (\( {\mathop V\limits^ \cdot }{\text{CO}}_{2} \)) were measured continuously during the final 20 min of both the basal state and during step 2 of the hyperinsulinaemic–euglycaemic clamp by indirect calorimetry using a ventilated hood system (Vmax model 2900; Sensormedics, Anaheim, CA, USA). The \( {\mathop V\limits^ \cdot }{\text{O}}_{2} \) and \( {\mathop V\limits^ \cdot }{\text{CO}}_{2} \) measurements during the last 10 min were used for further calculations.
Study medication
Prednisolone tablets were purchased from Pfizer (Sollentuna, Sweden) and matching placebo tablets were obtained from Xendo Drug Development (Groningen, the Netherlands). The tablets were encapsulated in order to allow the treatment to be blinded, as described previously [19]. Study medication was taken at 08:00 hours during the 2 week treatment except on days 13 and 14, when it was ingested at 06:00 hours. Patients kept a diary in which the exact time of medication intake during the study was registered.
Glucose, lipid, and valine measurements
Plasma glucose concentrations were measured with the glucose oxidase method using a Biosen C-line Plus glucose analyser (EKF Diagnostics, Barleben/Magdeburg, Germany). Plasma NEFA concentrations were measured with an enzymatic colorimetric method (NEFA-C test kit; Wako Chemicals, Neuss, Germany) with an intra-assay variation of 1%, inter-assay variation of 4–15% and a detection limit of 0.02 mmol/l. [6,6-2H2]Glucose, [1,1,2,3,3,-2H5]glycerol and L-[1-13C]valine enrichment were measured with gas chromatography–mass spectrometry as described previously [20–22]. Briefly, temperature-programmed gas chromatography with split injection (Model 6890; Agilent Technologies, Palo Alto, USA) was coupled to a mass selective detector (model 5973 Agilent Technologies) in the electron impact ionisation mode for glucose and valine analysis and in the positive chemical ionisation mode for glycerol. Exact gas chromatography and mass spectometry variables are given in Ackermans et al., Ackermans et al. and Geukers et al. [20–22].
Glucoregulatory hormones
Insulin was determined on an Immulite 2000 system (Diagnostic Products, Los Angeles, CA, USA) with a chemiluminescent immunometric assay, intra-assay variation of 3–6%, an inter-assay variation of 4–6% and a detection limit of 15 pmol/l. Glucagon was determined with the Linco 125I radioimmunoassay (St Charles, MO, USA) with an intra-assay variation of 9–10%, an inter-assay variation of 5–7% and a detection limit of 15 ng/l.
Calculations
Endogenous glucose production (EGP) and the peripheral uptake of glucose (rate of disappearance, R
d) were calculated using modified versions of the Steele equations for the non-steady state and were expressed as μmol kg−1 min−1 as described previously [23, 24]. Lipolysis (glycerol turnover) and proteolysis (valine turnover) were computed using formulae for steady state kinetics adapted for stable isotopes [20, 22] and were expressed as μmol kg−1 min−1. The equations are provided in ESM Fig. 3. The abbreviated Weir equation was used to calculate the 24 h energy expenditure. Glucose oxidation and fatty acid oxidation rates were derived from oxygen consumption and carbon oxide production as reported previously [25]. Glucose oxidation during insulin infusion was additionally expressed as percentage of glucose R
d.
Statistics
Data are presented as mean values ± SD, or as median (interquartile range) in case of skewed distribution. Absolute changes from baseline (on treatment value − pre-treatment value) were compared between the groups using the Kruskal–Wallis test. Non-parametric analysis was chosen because of the relatively small number of participants and the uneven group sizes. Only in the case of a significant finding were prednisolone 7.5 mg and prednisolone 30 mg compared against placebo by post hoc testing, using the Mann–Whitney U test. To correct for multiple testing, Bonferroni correction was applied by multiplying the obtained p value from the Mann–Whitney U test by the numbers of comparisons (i.e. two) that were carried out. All statistical analyses were run on SPSS for Windows version 15.0 (SPSS, Chicago, IL, USA). A p < 0.05 was considered statistically significant.