Study design and participants
This study was a randomized crossover trial that adhered to CONSORT guidelines [24]. The study adhered to the guidelines outlined in the Declaration of Helsinki, and the research protocol received approval from the institutional ethics committee of the National Institute of Fitness and Sports in Kanoya (approval number 22-1-5). The study was pre-registered with the University Hospital Medical Information Network Center (UMIN), a clinical trial registration system (ID: UMIN000048052). Enrollment began in April 2022 and ended in June 2022 through campus advertisements. The experiments, including preliminary testing and the main two trials, were conducted between July 2022 and September 2022. After providing a detailed explanation of the study’s purpose, design, protocol, and potential risks, each provided written informed consent from 13 young healthy men. Exclusion criteria included: (1) women (biological sex), (2) age < 18 or > 40 years, 2) regular exercise training habits, (3) consumption of medication known to affect lipid and carbohydrate metabolism, and (4) current smoking. Given the potential influence of hormonal fluctuations and changes in body composition due to the menstrual cycle in women, this study exclusively included men to minimize potential confounding factors in the results. The participant flow diagram is depicted in Fig. 1. The lead investigator enrolled the participants in the research and randomly assigned them to each experiment (carbohydrate ingestion + aerobic exercise (a carbohydrateingestion exercise (CE) trial) or fast + aerobic exercise (a fasted exercise (FE) trial)) using computer-generated random numbers (Microsoft Excel, Microsoft, USA).
Consolidated standards of reporting trials flow diagram for crossover trials. CE,a carbohydrate ingestion exercise trial; FE, a fasted exercise trial
Preliminary testing
Participants reported to our laboratory at least 1 week before the initial main experimental trial for baseline data assessment. Height was measured to the nearest 0.1 cm using a stadiometer. Weight, fat mass, fat-free mass, and skeletal muscle mass were measured to the nearest 0.1 kg using a dual-frequency body composition monitor (Inbody770; InBody Japan Inc., Tokyo, Japan). Body mass index was calculated as the weight in kilograms divided by the square of the height in meters. Blood pressure (systolic and diastolic) was measured using an automatic sphygmomanometer (HEM-1040, Omron Corp., Kyoto, Japan) after participants rested in a sitting position for 15 min. Aerobic capacity (VO2peak) was determined via an incremental exercise protocol with 15-watt (W) increases every 1 min after a brief warm-up period on a cycle ergometer (Aerobike 75XLIII, Konami Sports Life, Kanagawa, Japan). During the test, ventilation and gas exchange were measured using indirect calorimetry (K4b2, COSMED, Rome, Italy). The criteria for achieving VO2peak have been described previously [25]. The highest VO2 value achieved over 30 s was determined as the VO2peak.
Study protocol and procedures
The study protocol is shown in Fig. 2. The participants reported to our laboratory three times at intervals of at least one week to eliminate any potential carry-over effects. On the first day, they underwent preliminary testing (body composition, blood pressure, and aerobic capacity). On the remaining two days, they were randomly assigned to one of two experimental trials in a counterbalanced manner. The two experimental trials consisted of (1) CE trial and (2) FE trial. After an overnight fast for at least 12 h, participants arrived at our laboratory at 7:45 AM or 9:45 AM. The arrival time was set to the same time across the two trials for each participant. After each participant rested in the supine position for 10 min, a fasting blood sample was taken. In the CE trial, participants then ingested maltodextrin jelly (0.8 g/kg body weight) within 5 min. They then rested in a supine position on the bed for a further 30 min until the start of a cycling exercise. Participants ingested a further amount of maltodextrin jelly (0.4 g/kg body weight) at the start of the cycling exercise, comprising 40 min at a workload corresponding to 40% VO2peak. They ingested a further amount of maltodextrin jelly (0.4 g/kg body weight) 20 min into exercise. The FE trial had the same setting as the CE trial, except that participants remained fasted throughout the trial. After both exercises, participants rested in the supine position on a bed for 60 min and drank mineral water freely. The amount of carbohydrate intake to inhibit lipolysis was determined based on previous studies [22, 23]. Furthermore, the exercise intensity and duration were set based on a previous study where circulating FABP4 concentrations exhibited the highest reactivity to exercise [18]. Baseline and during aerobic exercise in the two trials, ventilation and gas exchange were measured using indirect calorimetry (K4b2, COSMED; Rome, Italy) at -5–0, 7–10, 17–20, 27–30, and 37–40 min from the start of exercise. Based on the mean of these data, energy expenditure (EE) and carbohydrate and fat oxidation rates were calculated from VO2, VCO2, and respiratory exchange ratio (RER) [26] every 10 min. Blood samples were collected from each participant at baseline, immediately before exercise, immediately after exercise, and 30 and 60 min post-exercise in both trials. Participants were instructed to consume the same meals for three days before each trial and to refrain from vigorous physical activity for 24 h before each trial.
Blood sampling and analysis
Blood samples were collected in 9-mL tubes containing thrombin and 7-mL and 2-mL tubes containing sodium EDTA. The 9-ml tubes were centrifuged at 3000 g for 10 min at room temperature after 30 min of collection. The 7-mL tubes were centrifuged at 3000 g for 10 min at 4 °C immediately after collection. The corresponding serum and plasma samples were transferred into plastic tubes and immediately stored at − 80 °C until further analysis. Blood in the 2-mL tubes was used to measure hemoglobin and hematocrit values to assess changes in plasma volume [27].
Venous blood samples were collected at baseline to determine plasma epinephrine and norepinephrine and serum insulin, total cholesterol (TC), high-density lipoprotein cholesterol (HDLC), triglyceride (TG), creatinine (Cre), glucose, free fatty acid (FFA), glycerol and FABP4 concentrations. Low-density lipoprotein cholesterol concentrations were estimated following Friedewald [28]. The remaining blood samples, except for the baseline blood samples, were used to determine plasma epinephrine and norepinephrine and serum insulin, glucose, FFA, glycerol, and FABP4 concentrations.
The plasma adrenaline and noradrenaline concentration were analyzed by high-performance liquid chromatography (Tosoh Corporation., Tokyo, Japan). The serum insulin concentration was measured via a chemiluminescent immunoassay (Abbott Japan, Tokyo, Japan). The serum TC and HDLC concentrations were measured via a direct enzymatic method (SEKISUI MEDICAL Corporation, Tokyo, Japan). The serum TG concentration was measured using the glycerol kinase–glycerol–3–phosphate oxidase erase method (Hitachi Chemical Diagnostics Systems Corporation., Tokyo, Japan). The serum Cre concentration was measured according to the endogenous creatine elimination reaction method (KANTO CHEMICAL Corporation, Tokyo, Japan). The estimated glomerular filtration rate (eGFR) was calculated using the equation for Japanese adult men (194 × creatinine concentration (mg/dl) −1.094 × age (years) −0.287). The serum glucose concentration was measured using an enzymatic method (Hitachi Chemical Diagnostics Systems Corporation., Tokyo, Japan). The serum TC concentration was measured using a cholesterol oxidase-peroxidase method (Hitachi Chemical Diagnostics Systems Corporation., Tokyo, Japan). The serum FFA concentration was measured using an enzymatic method (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan). The serum glycerol concentration was analyzed via a coupled enzymatic reaction (Cayman Chemical, MI, USA). The serum FABP4 concentration was measured using an enzyme-linked immunosorbent assay kit (R&D Systems Inc., MN, USA). To eliminate inter-assay variation, samples from each participant were analyzed in the same run. Intra-assay CV of analysis for FFA, glycerol and FABP4 concentration was < 5.0%.
Statistical analysis
The primary outcome was circulating FABP4 concentrations during the trials. Secondary outcomes included the effect of carbohydrate ingestion on circulating glycerol, FFA, insulin, and catecholamine concentrations. All data was obtained and there was no missing data. The Kolmogorov–Smirnov test and Levene’s test were used to confirm normality and homoscedasticity, respectively. In the case of non-normal distribution, a log transformation was performed. Glycerol, FFA, and FABP4 concentrations were statistically analyzed after log transformation. A paired-t test was used to determine differences in exercise intensity, EE, and substrate oxidation during exercise between the CE and FE trials. Two-way repeated-measures analysis of variance (trial × time) was used to compare changes in RER, and blood parameters between the two trials. When a significant interaction was observed, a Bonferroni post-hoc analysis was performed to determine differences between trials at a specific point in time. ES was calculated as Cohen’s d (small ≥ 0.20, medium ≥ 0.50, or large ≥ 0.80) for the post-hoc test. Pearson product-moment correlation coefficients were calculated to estimate the relationship between changes in glycerol and FFA levels during aerobic exercise (values after exercise– values before exercise, for ∆glycerol, and ∆FFA) and changes in circulating FABP4 concentrations following aerobic exercise (values after exercise– values 30 or 60 min after exercise, ∆30FABP4, and ∆60FABP4).
The sample size was calculated using the effect size (ES, f = 0.25) of the change in FABP4 concentration during exercise [18]. We determined that a sample size of at least nine would be required for approximately 80% power at 0.05 significance to detect an ES. The sample size was calculated using G*Power version 3.1.3 [29].
Statistical analyses were performed using SPSS version 28 software (IBM Corporation, Armonk, NY, USA). Data are presented as mean ± standard deviation (SD) or median (range). Values for blood parameters were adjusted according to plasma volume changes [27]. EE and carbohydrate and fat oxidation rates were calculated using VO2 and VCO2 [26]. Carbohydrate and fat oxidation rates were calculated using the following equations; Carbohydrate oxidation rate (g/min) = 4.585×VCO2 (L/min)– 3.226×VO2 (L/min), and Fat oxidation rate (g/min) = 1.695×VO2 (L/min)– 1.701×VCO2 (L/min). Statistical significance was set at p < 0.05.
