Materials and Methods
Patients with acromegaly were recruited through the outpatient clinic of the Department of Endocrinology at the Ghent University Hospital, Belgium. Inclusion criteria were as follows: age between 18 and 65 years, diagnosis of acromegaly since more than 1 year, treated by surgery, LA-SSA or combination of LA-SSA plus pegvisomant, no changes in treatment schedule during the last 6 months, serum IGF1 level below the age- and sex-specific threshold and a random GH <1.0 μg/L (for patients after surgery or under LA-SSA monotherapy). Exclusion criteria were as follows: liver function tests >3 times upper limit of normal, uncontrolled diabetes (glycated haemoglobin A1c [HbA1c] >60 mmol/mol [7.6%]), previous gallbladder resection, previous bariatric surgery, malabsorptive syndromes, hepatic or renal failure, concomitant use of insulin, sulfonylurea, fibrates or GLP-1 agonists, abuse of alcohol or drugs and body weight change of >10% during the last 12 months. The study protocol was approved by the Ethics Committee of the Ghent University Hospital and was pre-registered at clinicaltrials.gov (clinical trial registration number NCT02152124). All patients provided written informed consent before study participation.
Study Design and Study Protocol
Anthropometry and Whole-body Soft Tissue Composition. Participants had their body weight measured to the nearest 0.5 kg on a calibrated balance scale in light indoor clothing without shoes. Standing height was measured to the nearest 0.1 cm using a wall-mounted Harpenden stadiometer (Holtain Ltd.). Body mass index (BMI) was calculated as the weight (kg) divided by the height in metre squared (m2). Waist circumference was defined as the abdominal circumference at midway height between the lower rib margin and the iliac crest, hip circumference as the widest circumference around the buttocks. Waist-to-hip ratio (WHR) was calculated as the ratio of waist circumference over hip circumference. Whole-body soft tissue composition and lean body mass (LBM) were measured using dual-energy X-ray absorptiometry (Hologic QDR-4500A device, software version 11.2.1; Hologic).
Hyperinsulinaemic-euglycaemic Clamp Procedure. A hyperinsulinaemic-euglycaemic clamp procedure, conducted as proposed by Ferrannini and Mari, was performed to assess insulin sensitivity. The examination was initiated between 07.00 and 10.00 AM after an overnight fast. A primed-continuous insulin infusion was initiated and fixed at 40 mIU/m2 body surface area/min throughout the 120-min clamp to completely suppress endogenous glucose production. Variable infusion of glucose (200 g/L), adjusted every 5 minutes was used to maintain euglycaemia (90 mg/dl) during insulin infusion. In order not to underestimate arterial glucose levels, venous blood was arterialized through retrograde cannulation of a wrist or hand vein while heating the hand at 60–70°C using a custom-made heating box. During the clamp period, blood samples were drawn at 0, 30, 60, 90, 105 and 120 minutes. Steady state was defined as the last 30 minutes of the clamp period. The glucose disposal rate during this last half-hour corrected for LBM (M-value) was used as an estimate of insulin sensitivity and expressed in μmol/min/kg LBM.
Mixed Meal Test. To assess postprandial substrate metabolism and incretin response, a mixed meal test was performed. The administered meal was a liquid mixed meal (Boost Very High Calorie, Nestlé Health Science), providing a caloric content of 533 kcal (237 ml, 2.25 kcal/ml), whereby 50% of the energy came from fat, 34% from carbohydrates and 16% from proteins. Blood samples were taken before and 10, 30, 60, 120, 180, 240 and 300 minutes after ingestion. To minimize possible confounding effects of behavioural activities on the postprandial response, participants were asked to adhere to their standard lifestyle pattern regarding physical activity level and diet one week before the study visits.
Hormonal and Biochemical Assays. Serum was stored at −80°C until analysis; all samples from the same subject were assayed in a single assay run. IGF-1 was measured using the Immunodiagnostic Systems-iSYS IGF-I assay (IDS IGF-I) on the iSYS Multi-Discipline Automated System. Reference values were sex- and age-specific. Fasting triglycerides (TG), total cholesterol, high-density lipoprotein-cholesterol (HDL-C) and serum glucose concentrations were measured using standard laboratory assays (Cobas Modular Analyzer, Roche Diagnostics). Serum low-density lipoprotein-cholesterol (LDL-C) was calculated using Friedewald's formula (LDL-C (mg/dl) = total cholesterol (mg/dl)−HDL-C (mg/dl)−TG/5 (mg/dl)). Serum insulin levels were determined using the immunoanalyser COBAS e411 (Roche) which had an intra-coefficient of variation (intra-CV) between 2.93% (μ = 14.16 μU) and 2.38% (μ = 62.6 μU). HbA1c was analysed using the Tosoh Automated Glycohemoglobin Analyzer HLC-723G8 (G8) analyzer and reported in % of glycosylated HbA1c. GIP and GLP-1 were measured at the Copenhagen Novo Nordisk Foundation Center for Basic Metabolic Research. GIP and GLP-1 concentrations in plasma were measured after extraction of plasma with 70% ethanol (vol/vol, final concentration). For the GIP radioimmunoassay, we used the C-terminally directed antiserum code # 867, which was raised against a synthetic peptide corresponding to the C-terminus of human GIP. It does not cross-react with the so-called GIP 8000 of which chemical nature and relationship to GIP secretion is uncertain. It reacts fully with the primary metabolite, GIP 3–42. Human GIP and 125-I human GIP (70 MBq/nmol) were used for standards and tracer. The plasma concentrations of GLP-1 were measured against standards of synthetic GLP-1 7–36amide using antiserum code no. 89390, which is specific for the amidated C-terminus of GLP-1 and therefore does not react with GLP-1-containing peptides from the pancreas. The results of the assay accurately reflect the rate of secretion of GLP-1 because the assay measures the sum of intact GLP-1 and the primary metabolite, GLP-1 9–36amide, into which GLP-1 is rapidly converted. For both assays, sensitivity was below 1 pmol/L, intra-assay coefficient of variation was below 6% at 20 pmol/L, and recovery of standard, added to plasma before extraction, was about 100% when corrected for losses inherent in the plasma extraction procedure.
Data Analysis. Variables were checked for normality using the Shapiro-Wilk test. Log-transformation was used to normalize skewed data. Area under the curve (AUC) was calculated using the linear trapezoidal method (AUC = (C1 + C2) × (t2−t1)/2). Unless otherwise stated, variables were expressed as median (interquartile range). Correlations between variables were analysed using Spearman's rank-order correlation reported as Spearman's correlation coefficient rs. Differences between the three groups were explored using an analysis of variance (ANOVA) for continuous variables and chi-square test for nominal variables. Correction for covariates was performed by analysis of covariance (ANCOVA). p < .05 were considered statistically significant. All p-values were two-tailed. Statistical procedures were performed using SPSS 25.0 software package (SPSS Inc).
Clin Endocrinol. 2021;95(1):65-73. © 2021 Blackwell Publishing