Subject
A healthy male Caucasian (age: 53 years; body weight: 90.7 kg; body mass index: 28.6 kg/m2) with normal glucose homeostasis (fasting plasma glucose: 5.9 mM; 2 h plasma glucose value following 75 g-oral glucose tolerance test: 3.5 mM; glycated haemoglobin A1c: 5.5%) and without any family history of diabetes was examined. Insulin resistance according to the homeostasis model assessment [8] was 1.65. He had no significant past medical history, took no medication and clinical examination was normal. All standard investigations including blood pressure and standard biochemical parameters in urine and blood were normal. He agreed to participate (verbal and written consent) after receiving oral and written information. The examinations were approved by the Scientific-Ethical Committee of the County of Copenhagen (journal no.: KA 97196 m) and conducted according to the principles of the Helsinki Declaration II.
Methods
The subject participated in a study evaluating the insulinotropic power of supra-physiological doses of GLP-1 as compared to glucagon, GIP, and physiological conditions in healthy subjects [9]. He was examined on 4 different days (day 1 through 4) separated by minimum 48 hours. On each occasion the subject was studied after a 10 h-fast, including liquids, with a cannula inserted in a dorsal hand vein for collection of blood samples. On day 2, 3 and 4, another cannula was inserted into the contra-lateral cubital vein for infusion of glucagon (day 2) and glucose plus incretin hormones (day 3 and 4).
Meal test (day 1)
Blood was drawn regularly after a standard breakfast meal ingested during 15 min. The meal comprised 2,370 kJ (566 kcal) and was composed of 34% fat, 47% carbohydrate, and 19% protein.
Glucagon test (day 2)
Blood was sampled 15, 10 and 0 min before and 2, 3, 4, 6, 8, 10, 15, 20, 30, 45 min after iv bolus of 1 mg (=287 nmol) biosynthetic glucagon (GlucaGen, Novo Nordisk, Bagsværd, Denmark) in 1 ml of sterile water.
Hyperglycaemic clamps + GLP-1 (day 3) or GIP (day 4)
At time 0 min, a bolus of 50% glucose (w/v) was infused during 1 min to increase plasma glucose to 15 mM. Plasma glucose was kept at 15 mM by continuous infusion of glucose, which was adjusted every 5 min according to bedside measurements of plasma glucose. After 3 min, a continuous infusion of 1 pmol GLP-1/kg/min (day 3) or 4 pmol GIP/kg/min (day 4) was initiated. Blood was sampled 15, 10 and 0 min before and 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105 and 120 min after elevation of plasma glucose.
An additional experimental day consisting of a 2 h 15 mM-hyperglycaemic clamp with continuous infusion of saline was designed in order to evaluate the insulin response to hyperglycaemia without incretin hormone infusion. However, the subject withdrew his consent due to the symptoms of hypoglycaemia experienced following termination of the hyperglycaemic clamp with infusion of GLP-1 (see below). Thus, we found it unethical to utilize the present design in further evaluations of beta cell function in healthy subjects [9].
On all experimental days, blood was sampled into tubes containing heparin or EDTA (6 mM) plus aprotinin (500 KIU/ml blood; Trasylol, Bayer, Leverkusen, Germany) and a specific dipeptidyl peptidase 4 (DPP-4) inhibitor (valine-pyrrolidide; 0.01 mM, final concentration; a gift from Novo Nordisk A/S Bagsværd, Denmark) for hormone analyses. These tubes were immediately cooled on ice and centrifuged for 20 min at 1,200 g and 4°C. Plasma was stored at -20°C until analysis. For bedside measurements of plasma glucose, blood was distributed into fluoride tubes and centrifuged immediately for 2 min at 7,400 g and room temperature.
Analyses
Plasma glucose concentrations were measured during the experiments by the glucose oxidase method (Yellow Springs Instrument Model YSI 2300 STAT plus analyzer; YSI Inc., Yellow Springs, Ohio, USA).
Plasma samples were assayed for total GLP-1 immunoreactivity using antiserum no. 89390, which is specific for the C-terminal of the GLP-1 molecule and reacts equally with intact GLP-1 and the primary (N-terminally truncated) metabolite.
Total GIP was measured using the C-terminally directed antiserum R65, which reacts fully with intact GIP and the N-terminally truncated metabolite.
Plasma insulin concentrations were measured using commercial ELISA kits (Dako, Copenhagen, Denmark).
Plasma C-peptide concentrations were determined as described by Heding et al.[10] employing the polyclonal antibody M1230.
Calculations and statistics
Area under the curve (AUC) values were calculated using the trapezoidal rule and data is presented using standard descriptive statistics.