The study on lifestyle intervention and IGT, Maastricht, was designed to study whether a diet/physical activity intervention program can improve glucose tolerance in subjects with a high risk of developing type 2 diabetes. A detailed description of the study can be found elsewhere (18). In the present study, 16 subjects with IGT [nine intervention (INT) subjects, seven controls (CON)] participating in the larger intervention trial underwent additional measurements to evaluate the effect of this lifestyle intervention program on substrate use and oxidation (for characteristics, see Table 1). Subjects had no other reported health problems and did not use any medication that could interfere with substrate metabolism. The Medical Ethical Review Committee of Maastricht University approved the study protocol, and all subjects gave their written informed consent before the start of the study.
For a detailed description, see reference (18). Briefly, the intervention program consisted of a dietary and a physical activity component. Dietary recommendations were based on the Dutch guidelines for a healthy diet (±55 energy% carbohydrates; <30 to 35 energy% fat intake, with <10 energy% intake of saturated FAs; a cholesterol intake of <33 mg/MJ; protein intake of 10 to 15 energy%; and an intake of dietary fiber of at least 3 g/MJ). Dietary advice was given by a skilled dietitian on an individual basis every 3 months. Subjects were stimulated to increase their physical activity to at least 30 minutes of moderate physical activity a day for at least 5 d/wk. Individual advice was given on how to increase their daily physical activity (walking, cycling, swimming). Furthermore, subjects were encouraged to participate in an exercise program consisting of components of aerobic exercise training and components of resistance training.
Subjects in the CON group were informed about the beneficial effects of a healthy diet, weight loss, and increased physical activity, but no individual advice or programs were provided. No additional appointments were scheduled.
Before and after 1 year of the lifestyle intervention program, several measurements were performed.
Glucose Tolerance Test
A standard oral glucose tolerance test (OGTT), with blood sampling at t = 0, 30, 60, and 120 minutes, was performed to measure glucose tolerance. Fasting plasma glucose (millimolar) and insulin concentration (milliunits per liter) were used to calculate an index for insulin resistance with the homeostasis model assessment (HOMA-IR) described by Matthews et al. (19).
An incremental exhaustive exercise test was performed on an electronically braked bicycle ergometer (Lode, Groningen, The Netherlands) to determine the maximal aerobic power output and oxygen consumption (Vo2max). The test started at a workload of 0.75 W/kg fat-free mass (FFM) for 3 minutes, followed by 3 minutes at 1.5 W/kg FFM. Thereafter, the workload was increased every 3 minutes by 0.5 W/kg FFM until exhaustion (respiratory quotient above 1.1 and no further increase in oxygen uptake). During the experimental trials, subjects exercised before and after 1 year at the same absolute workload, i.e., 55% baseline Vo2max.
Body weight (BW) was determined on an electronic scale; body composition was determined by hydrostatic weighing with simultaneous lung volume measurement (Volugraph 2000; Mijnhardt, Bunnik, The Netherlands), and calculated according to Siri (20). Waist and hip circumference measurements were made to the nearest 1 cm with subjects standing in an upright position, one-half way between the iliac spine and the last rib and at the level of the trochanter major, respectively.
Subjects participated before and after 1 year in two stable-isotope trials, separated by at least 1 week. Trials were performed in random order. Subjects were asked not to participate in any (exhausting) physical activity the last 3 days before the trials and not to consume any products of high natural 13C-abundance during the last week before both tests because this may disturb the 13C/12C measurement in blood and expired air (21).
Subjects came to the laboratory at 8 am after an overnight fast. Two cannulae were inserted, one into an antecubital vein for the infusion of tracers and one in retrograde direction into a contralateral dorsal hand vein for blood sampling. The cannulated hand was placed in a hot box to obtain arterialized venous blood. Background blood and breath samples were taken 30 minutes after placement of the cannulae. At t = 0, an intravenous dose of 0.085 mg/kg BW NaH13CO3 was given to prime the bicarbonate pool, followed by a constant rate continuous infusion of [U-13C]palmitate (0.0067 μmol/kg BW per minute). After 60 minutes, a continuous infusion of [6, 6-2H2]glucose (0.3 μmol/kg BW per minute) was started after a priming dose had been given (18 μmol/kg BW per minute). Tracers were administered through a calibrated infusion pump (IVAC560 pump; IVAC, San Diego, CA). During the last 20 minutes of the resting period (t = 100, 110, and 120 minutes), breath and blood samples were taken, and Vo2 consumption and vco2 production were determined. Thereafter, exercise was started for 1 hour (120 to 180 minutes). [U-13C]palmitate infusion was doubled at the start of the exercise. During the last 20 minutes, blood and breath samples were taken, and Vo2 consumption and vco2 production were determined (t = 160, 170, and 180 minutes).
In a second trial, the acetate recovery factor (ARF), necessary for correction of palmitate oxidation rates (22), was determined as described before (11)
The [U-13C]palmitate tracer (99% enriched; Cambridge Isotope laboratories, Andover, MA) was dissolved in heated sterile water and passed through a 0.2-μm filter into 5% warm human serum albumin (Central Blood Bank, Leiden, The Netherlands) to make a 0.65 mM solution. The [6, 6-2H2]glucose and [1, 2-13C]acetate tracer (99% enriched, Cambridge Isotope Laboratories) were dissolved in 0.9% saline to make an 18.7 and 3.0 mM solution, respectively. The exact infusion rates of [U-13C]palmitate, [6, 6-2H2]glucose, and [1, 2-13C]acetate were determined for each experiment by measuring the concentration of the infusate (see “Biochemical Methods”).
Breath, Blood, and Urine Sampling
Breath samples were obtained by having the subjects breathe normally for at least 3 minutes into a mouthpiece connected to a 6.75-liter mixing chamber. Breath samples were collected into a 20-mL Vacutainer tube (Becton Dickinson, Meyland Cedex, France) to determine the enrichment of CO2 (13C/12C ratio). Vo2 and vco2 were determined by means of open-circuit spirometry (Oxycon Beta; Mijnhardt). Arterialized blood samples were collected in EDTA-containing tubes and were immediately centrifuged at 3000 rpm at 4 °C; the plasma was frozen in liquid nitrogen and stored at −80 °C until analysis. Urine was collected overnight to determine nitrogen excretion for calculating the non-protein respiratory exchange ratio.
Breath samples were analyzed for 13C/12C ratio by injecting 20 μL of the gaseous headspace into a gas chromatograph (GC)-isotope ratio mass spectrometer (Finnigan MAT 252; Finnigan, Bremen, Germany). Total plasma FFA, glucose, and infusate acetate concentrations were measured using standard enzymatic techniques (for FFA, FFA-C test kit, Wako Chemicals, Neuss, Germany; for glucose, Roche Unikit III, Hoffman-La Roche, Basel, Switzerland; for acetate, kit no. 148261, Boehringer Mannheim, Mannheim, Germany). Insulin concentration during the experimental trial was measured using a double-antibody radioimmunoassay (Insulin RIA-100; Kabi Pharmacia, Uppsala, Sweden); plasma insulin levels during the OGTT were measured with an enzyme-linked immunosorbent assay (Mercodia, Uppsala, Sweden). For the determination of plasma palmitate concentration and enrichment, FFAs were extracted from plasma, isolated by thin-layer chromatography, and derivatized to their methyl esters. Palmitate concentrations were determined on an analytical GC with ion-flame detection using heptadecanoic acid as an internal standard; on average, palmitate concentration was 27 ± 1% of total FFA concentration. Isotopic enrichment of palmitate was determined by GC-isotope ratio mass spectrometer after on-line combustion of FAs to CO2 (Finnigan MAT 252), with correction for the extra methyl group in the derivate. The concentration of infusate palmitate was determined as described above for plasma samples. For determination of glucose enrichment in plasma, aliquots of EDTA plasma were extracted with methanol:chloroform and chloroform:water. The clear water layer was dried, and a butylboronic acid-acetyl derivate was made. Subsequently, the enrichment of the glucose derivate was determined by electron ionization/gas chromatography mass spectrometry (Finnigan INCOS XL; Finnigan, San Jose, CA).
Metabolic rate was calculated from Vo2 and vco2 according to the equation of Weir (23). Carbohydrate and fat oxidation rates were calculated from Vo2 and vco2 and urinary nitrogen excretion (24). Protein oxidation (as calculated from nitrogen excretion) was assumed to be similar during the overnight fasted state and during exercise. Total FA oxidation was calculated by converting the rate of fat oxidation [triglyceride (TG) oxidation] to its molecular equivalent, with the assumption of the average molecular weight of TG to be 860 g/mol. Subsequently, the molar rate of TG oxidation was multiplied by three to obtain FA oxidation.
Enrichment of breath CO2 and plasma palmitate, acetate, and glucose is given as the tracer-to-tracee ratio [TTR; =(13C/12C)sample − (13C/12C)background]. Fractional recovery of label in breath CO2, derived from the infusion of labeled acetate, was calculated as follows: acetate recovery = (TTRCO2 × vco2)/2F, where F is the infusion rate of acetate, and the number 2 in the denominator is to correct for the number of 13C molecules in acetate.
Rate of appearance (Ra) and rate of disappearance (Rd) were calculated according to Steele's equation for steady state (palmitate at rest) and Steele's single-pool non-steady state equations adapted for use with stable isotopes (palmitate during exercise and glucose at rest and during exercise). Volume of distribution was assumed to be 0.040 L/kg for palmitate and 0.160 L/kg for glucose. Ra and Rd of FFA were calculated by dividing palmitate Ra and Rd by the fractional contribution of palmitate to the total FFA concentration. Percentage of infused [U-13C]palmitate oxidized was calculated with the formula: percentage infused tracer oxidized = ((TTR CO2× vco2)/(16 × F × acetate recovery)) × 100%, where F is the infusion rate of palmitate, and the number 16 in the denominator is to correct for the number of 13C molecules in palmitate. Plasma FFA oxidation was calculated as: Rd FFA × percentage of infused palmitate tracer oxidized, and TG-derived FA oxidation was calculated as: total FA oxidation − plasma FFA oxidation. During exercise, the Rd of glucose is identical to the measured oxidation rate (25); therefore, muscle glycogen oxidation rates during exercise are calculated as: total glucose oxidation − plasma glucose oxidation.
Data are presented as means ± SE. Oxidation rates and Ra and Rd are expressed as micromoles per kilogram of FFM per minute. Differences between groups were analyzed with a two-tailed Student's t test for unpaired data, and changes within groups were analyzed with a two-tailed Student's t test for paired data. Changes in concentration of metabolites over time between groups were analyzed with a two-way repeated-measures ANOVA. Statistical significance was set at p < 0.05.