Efficacy of a Nutritional Education Strategy and Role of Physical Exercise on the Regulation of Appetite and Body Composition Through the Profile of Exosomes in Type 2 Diabetics (The APETEX Project)
试验速览
- 阶段
- 不适用
- 状态
- 已完成
- 入组人数
- 146
- 试验地点
- 2
- 主要终点
- Assessed changes in Plasma Exosomal miRNA expression
研究概览
简要总结
The aim of this randomized controlled trial is to investigate the effects of different aerobic modalities: moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT), and a nutritional intervention (D) on appetite regulation, appetite-related hormones, neural responses to food cues (EEG), and exosome-derived molecular signals in patients with prediabetes or type 2 diabetes (T2D), and overweight or obesity. It will also examine whether these effects differ between men and women.
The main questions it aims to answer are:
- What is the isolated and combined effect of these exercise modalities and diet on glucose metabolism, insulin dynamics, miRNA exosomal expression, appetite hormones, and neural responses to food stimuli?
- Is there a sex-specific best combination choice for improving appetite regulation and metabolic control?
- Are changes in miRNA exosomal profile, appetite-related hormones, and EEG-based neural responses associated with other health-related outcomes such as body composition, fat mass reduction, insulin sensitivity, physical condition, and quality of life?
To answer these questions, researchers designed a two-factor study. One factor was diet, with two levels (Diet (D) and no diet (ND)). The second factor was exercise, with three levels (inactive (INACT), MICT, and HIIT). Participants were randomly allocated into six groups: ND-INACT, ND-MICT, ND-HIIT, D-INACT, D-MICT, D-HIIT.
Participants did:
- Underwent a 12-week intervention within their assigned group.
- Visited the research facilities three times per week if assigned to an exercise group, or once every two weeks if in a diet group.
详细描述
This randomized controlled trial was conducted in the Province of Cádiz (Spain). A total of 146 participants were enrolled in the study and underwent baseline assessments of the study outcomes. Immediately after baseline evaluations, participants were randomly allocated to one of the six study groups. Randomization was stratified by sex to ensure approximately 50% of participants in each group were women. All participants received detailed written and verbal information about the study objectives, procedures, benefits, and potential risks before providing written informed consent. The study complied with the Declaration of Helsinki and was approved by the Research Ethics Committee of Cádiz (registration number 92.21; PEIBA number 1026-N-21) and by the Coordinating Committee for Biomedical Research Ethics of Andalusia (registration number 92.21; PEIBA number 1026-N-21; internal codes SICEIA-2025-001870 and SICEIA-2025-000778). Sample size was calculated a priori using G*Power (v3.1.9.7; Universität Düsseldorf, Germany) based on repeated-measures ANOVA (within-between interaction). Targeting a conservative small-to medium effect size (f=0.19). Assuming an alpha level of 0.05 and a statistical power of 0.90 across the six study subgroups, 120 participants were required. Accounting for ~20% attrition, 146 were recruited.
At both baseline and post-intervention, participants were scheduled in the morning following an overnight fast of 8-10 hours. They were instructed to maintain their usual lifestyle, abstain from alcohol and caffeine for 24 hours, and avoid strenuous physical activity for at least 48 hours prior to testing. During these visits, a comprehensive set of assessments was conducted to capture the effects of the interventions across multiple domains. These included measures of glucose metabolism, insulin dynamics, plasma exosomal miRNA expression, appetite-regulating hormones, neural responses to food cues, subjective appetite, body composition, dietary intake and adherence, physical activity and sedentary behavior, resting and maximal fat oxidation, cardiorespiratory fitness, blood pressure, and self-reported quality of life. Post-intervention assessments followed the same protocol, with participants in exercise groups completing their final training session at least 48 hours before testing (range 48 - 72 hours). This design allows evaluation of both the isolated and combined effects of exercise and diet interventions on physiological, molecular, behavioral, and lifestyle outcomes, as well as potential sex-specific differences in response.
研究设计
- 研究类型
- Interventional
- 分配方式
- Randomized
- 干预模型
- Parallel
- 主要目的
- Treatment
- 盲法
- Double (Investigator, Outcomes Assessor)
盲法说明
Due to the nature of the intervention, participants and intervention providers can not be blinded. However, outcome assessors and statisticians remained blinded to group allocation. Additionally, the randomization procedure was administered by an independent researcher.
入排标准
- 年龄范围
- 30 Years 至 65 Years（Adult, Older Adult）
- 性别
- All
- 接受健康志愿者
- 否
入选标准
- •Diagnosis of type 2 diabetes or prediabetes (HbA1c ≥ 5.7%)
- •No history of substance abuse (tobacco, alcohol, or other drugs).
- •Body mass index ≥25 kg/m², maintaining habitual dietary patterns with stable body weight for at least 6 months
- •Stable physical activity and medication regimen for at least 6 months
- •Lack of habitual vigorous-intensity physical activity and the presence of sedentary behavior characterized by prolonged sitting time (screening using the International Physical Activity Questionnaire short form).
- •Not insulin-dependent
- •Absence of injury, disease, disability, or other known medical condition which could affect the ability to successfully participate in physical exercise tests
- •Absence of tumors and cancer disease
- •Absence of other pathologies that could affect the study outcomes.
- •Not under medication regimen that could affect the study outcomes.
- •Being able to understand communication in Spanish or English.
- •Not pregnant
排除标准
- •Failure to attend more than 2 consecutive sessions or a total of 4 sessions of nutritional intervention.
- •Failure to attend more than 4 consecutive or a total of 6 sessions of physical training.
- •Serious injury
- •Voluntary withdrawal
- •Increased dose of diabetes medication
研究组 & 干预措施
ND-INACT
Participants who did not receive either nutritional intervention or an exercise program. They were instructed to maintain their normal life habits with respect to physical activity and diet.
Nutritional Intervention High-intensity interval training (D-HIIT)
Participants who received nutritional intervention and were enrolled in a high-intensity interval training exercise program.
干预措施: Nutritional Intervention (Behavioral)
Moderate-intensity continuous training (ND-MICT)
Participants who did not receive nutritional intervention but were enrolled in a moderate-intensity continuous training exercise program.
干预措施: Moderate-intensity continuous training (MICT) (Other)
High-intensity interval training (ND-HIIT)
Participants who did not receive nutritional intervention but were enrolled in a high-intensity interval training exercise program.
干预措施: High-intensity interval training (HIIT) (Other)
Nutritional Intervention (D-INACT)
Participants who received nutritional intervention but not an exercise program.
干预措施: Nutritional Intervention (Behavioral)
Nutritional Intervention Moderate-intensity continuous training (D-MICT)
Participants who received nutritional intervention and were enrolled in a moderate-intensity continuous training exercise program.
干预措施: Moderate-intensity continuous training (MICT) (Other)
Nutritional Intervention Moderate-intensity continuous training (D-MICT)
Participants who received nutritional intervention and were enrolled in a moderate-intensity continuous training exercise program.
干预措施: Nutritional Intervention (Behavioral)
Nutritional Intervention High-intensity interval training (D-HIIT)
Participants who received nutritional intervention and were enrolled in a high-intensity interval training exercise program.
干预措施: High-intensity interval training (HIIT) (Other)
结局指标
主要结局
Assessed changes in Plasma Exosomal miRNA expression
时间窗: Pre and Post the 12-week intervention
Fasting blood samples (fast 8-10 hours) were centrifuged (1500×g, 15 min, 4 °C) and the resulting plasma was divided into aliquots (500 µL) and stored at -80°C. Exosomes will be isolated from 600 µL of plasma using the miRCURY Exosome Kits (Qiagen, Netherlands). Total RNA was extracted from 200 μL of exosome samples using the miRNeasy Serum/Plasma Advanced Kit (Qiagen). Reverse transcription was performed with the miRCURY LNA RT Kit, incubated at 42°C for 60 min, 95°C for 5 min, and stored at -20°C. Quantitative PCR was carried out using the miRCURY LNA SYBR Green PCR Kit and miRNA-specific assays on a CFX Connect System (Bio-Rad). MiRNA expression was normalized to validated endogenous reference miRNAs, and relative expression and pre-to-post fold-changes were calculated to ensure accurate quantification of exosomal miRNA expression.
Glycated hemoglobin
时间窗: Pre and Post the 12-week Intervention
Glycated hemoglobin (HbA1c, %) was measured using high-performance liquid chromatography (HPLC) with the automated ADAMS™ HA-8180V analyzer (ARKRAY, Kyoto, Japan).
Assessed changes in Anthropometry: Skinfold thickness
时间窗: Pre and Post 12-week Intervention
Thigh skinfold thickness was measured at two-thirds of the distance from the iliospinale point (the lowest part of the anterior superior iliac spine) to the superolateral border of the patella, over the belly of the vastus lateralis muscle. Measurements were taken on the dominant leg. Two measurements were obtained, and a third was taken if the difference between the first two exceeded 5%. The mean of the two most similar records was recorded.
Assessed changes in Glucose concentration
时间窗: Pre and Post the 12-week intervention
Glucose concentration (mg/dL) was measured from blood samples collected from the antecubital vein after at least 8 hours of fasting and at 30, 60, 90, 120, and 180 minutes after the ingestion of 75 grams of glucose (Nuter-tec, 75 g/200 mL, orange flavor). Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine consumption for 24 hours, and avoid vigorous physical activity for 72 hours. Blood samples were centrifuged at 4000 rpm for 7 minutes, and serum was analyzed. Glucose concentrations were determined using the colorimetric method performed on the Alinity CI system (Abbott Laboratories, Abbott Park, IL, USA).
Assessed changes in Insulin concentration
时间窗: Pre and Post the 12-week intervention
Insulin concentration (µU/mL) was measured from blood samples collected from the antecubital vein into serum separator tubes after at least 8 hours of fasting and at 30, 60, 90, 120, and 180 minutes after the ingestion of 75 grams of glucose (Nuter-tec, 75 g/200 mL, orange flavor). Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine consumption for 24 hours, and avoid vigorous physical activity for 72 hours. Blood samples were centrifuged at 4000 rpm for 7 minutes, and serum was analyzed. Insulin concentrations were determined by chemiluminescent immunoassays performed on the Alinity CI system (Abbott Laboratories, Abbott Park, IL, USA).
Assessed changes in Insulin Resistance: HOMA-IR
时间窗: Pre and Post 12-week intervention
Insulin resistance was estimated using the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), calculated as fasting insulin (µU/mL) multiplied by fasting glucose (mg/dL) divided by 405.
Assessed changes in Insulin Sensitivity: Matsuda Index
时间窗: Pre and Post 12-week intervention
Whole body insulin sensitivity was estimated through the Matsuda Index, calculated as 10,000 / √\[(fasting glucose (mg/dL) × fasting insulin (µU/mL)) × (mean glucose during Oral Glucose Tolerance Test (mg/dL) × mean insulin during Oral Glucose Tolerance Test (µU/mL))\].
Assessed changes in Insulin Sensitivity: Quicki
时间窗: Pre and Post 12-week intervention
Insulin sensitivity was estimated through the Quantitative Insulin Sensitivity Check Index (QUICKI), calculated as 1 / \[log(fasting insulin (µU/mL)) + log(fasting glucose (mg/dL))\].
Assessed changes in Anthropometry: Perimeters
时间窗: Pre and Post 12-week Intervention
Waist circumference (cm) was measured at the midpoint between the lower lateral margin of the 10th rib and the iliac crest, perpendicular to the longitudinal axis of the trunk. Abdominal circumference (cm) was measured at the level of the umbilicus, perpendicular to the longitudinal axis of the trunk. Hip circumference (cm) was measured at the level of the greatest posterior prominence of the buttocks, perpendicular to the longitudinal axis of the trunk. Thigh circumference (cm) was measured at the midpoint between the greater trochanter and the lateral tibial condyle. All measurements were performed with the participant standing upright, and relaxed. Each measurement was taken twice, and a third measurement was performed if the difference exceeded 1%. The mean of the two most similar records was registered.
Assessed changes in Appetite hormones
时间窗: Pre and Post the 12-week intervention
Appetite-regulating hormones will be assessed from blood samples collected after an 8-10-hour overnight fast, with participants seated at rest for 15 minutes. Fasting samples will be drawn from the antecubital vein using EDTA tubes for appetite hormones and CAT tubes for serum cortisol. EDTA tubes will be inverted, centrifuged at 1500 × g for 15 minutes at 4 °C, and plasma aliquoted into 500 µL tubes with 5 µL protease inhibitors, then stored at -80 °C. Ghrelin, leptin, GLP-1, and GIP will be measured fasting and 90 minutes post-75 g glucose using the MILLIPLEX® Metabolic Hormone Panel V3 with a Luminex® xMap system; intra- and inter-assay CVs \<10% and \<20%. Cortisol from CAT tubes allowed to clot 30 minutes, centrifuged at 2000 × g for 10 minutes at 4 °C, aliquoted into 200 µL tubes, stored at -80 °C, and quantified using one-step CMIA on Alinity CI; emitted light inversely reflects cortisol, range 1-59.8 µg/dL, intra- and inter-assay variation \<10%.
Assessed changes in task of emotional reactivity to food images
时间窗: Pre and Post the 12-week intervention
Emotional reactivity to food images will be measured using a high-density 128-channel EEG system (HydroCel Sensor Net, EGI, Inc.) during both resting state and a visual-attentional cognitive task. EEG caps will be fitted according to standardized landmarks (GND at the forehead, REF at the vertex, lateral electrodes at the eyebrows and top of ears, posterior electrode at the occipital bone), with impedance checked and adjusted by pipetting or gentle repositioning. Participants will be seated 60-70 cm from a 15'' monitor in dim lighting, with eyes aligned to the center of the screen. A 4-minute eyes-open resting-state recording will be conducted to assess baseline alpha activity (8-14 Hz). The experimental task will present images in blocks with breaks, including high- and low-calorie foods, non-food controls, and, in a separate task, images categorized as beneficial or harmful for gut microbiota based on fiber, prebiotic/probiotic content, caloric density, fat, and meat content.
Assessed changes in Appetite assessment.
时间窗: Pre and Post the 12-week intervention
Since appetite feelings modulate nutritional behavior, evaluating appetite among the intervention groups can improve the study's quality from a comprehensive perspective. After an 8-10 hour fasting period, a Visual Analogue Scale (VAS) will be completed to measure the participant's morning appetite. Likewise, they will be evaluated 90 minutes into the Oral Glucose Tolerance Test (OGTT) and at the end of it. The VAS is a valid measurement instrument for assessing the feeling of appetite.
Assessed changes in Body Weight
时间窗: Pre and Post the 12-week intervention
Body weight (kilograms, kg) was measured using a multifrequency bioelectrical impedance device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for 72 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.
Assessed in Height
时间窗: Pre and Post 12-week Intervention
Height (meters, m) was measured using a wall-mounted stadiometer (TANITA-LEICESTER HR-001, Tanita Corp., Tokyo, Japan) with participants standing in accordance with the manufacturer's instructions.
Assessed changes in Body Mass Index
时间窗: Pre and Post 12-week Intervention
Body Mass Index (BMI) in kilograms per square meter (kg/m²) was calculated as body weight (kg) divided by height squared (m²).
Assessed changes in Fat Mass (kilograms of Fat Mass)
时间窗: Pre and Post 12-week Intervention
Fat mass (kilograms, kg) was estimated using multifrequency bioelectrical impedance with an 8-electrode device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for 72 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.
Assessed changes in Fat Mass (percentage of Fat Mass)
时间窗: Pre and Post 12-week Intervention
Fat mass (percentage of total body weight, %) was estimated using multifrequency bioelectrical impedance with an 8-electrode device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for 72 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.
Assessed changes in Fat-free Mass (kilograms of Fat-free Mass)
时间窗: Pre and Post 12-week Intervention
Fat-free mass (kilograms, kg) was estimated using multifrequency bioelectrical impedance with an 8-electrode device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for 72 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.
Assessed changes in Fat-free Mass (percentage of Fat-free Mass)
时间窗: Pre and Post 12-week Intervention
Fat-free mass (percentage of total body weight, %) was estimated using multifrequency bioelectrical impedance with an 8-electrode device (TANITA-MC780MA, Tanita Corp., Tokyo, Japan) after at least 8 hours of fasting. Participants were instructed the day before testing to maintain their usual lifestyle, refrain from alcohol or caffeine for 24 hours, and avoid vigorous physical activity for 72 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.
Assessed changes in Anthropometry: Waist to Hip Ratio
时间窗: Pre and Post 12-week Intervention
Waist-to-hip ratio (WHR) was calculated as waist circumference (cm) divided by hip circumference (cm).
次要结局
- Assessed changes in dietary intakes: Mediterranean Diet Adherence(Pre and post the 12-week intervention)
- Assessed changes in accelerometry: Physical activity time(Pre and post the 12-week intervention)
- Assessed changes in Resting Energy Expenditure(Pre and post the 12-week intervention)
- Assessed changes in self-reported quality of life(Pre and post the 12-week intervention)
- Assessed changes in blood pressure(Pre and post the 12-week intervention)
- Assessed changes in Glucose concentration(Pre and Post the 12-week intervention)
- Assessed changes in Insulin concentration(Pre and Post the 12-week intervention)
- Assessed changes in Appetite assessment.(Pre and Post the 12-week intervention)
- Assessed changes in task of emotional reactivity to food images(Pre and Post the 12-week intervention)
- Assessed changes in Appetite hormones(Pre and Post the 12-week intervention)
- Assessed changes in Plasma Exosomal miRNA expression(Pre and Post the 12-week intervention)
- Assessed changes in Insulin Sensitivity: Quicki(Pre and Post 12-week intervention)
- Assessed changes in Insulin Sensitivity: Matsuda Index(Pre and Post 12-week intervention)
- Assessed changes in Insulin Resistance: HOMA-IR(Pre and Post 12-week intervention)
- Assessed changes in Anthropometry: Skinfold thickness(Pre and Post 12-week Intervention)
- Assessed changes in Anthropometry: Waist to Hip Ratio(Pre and Post 12-week Intervention)
- Assessed changes in Anthropometry: Perimeters(Pre and Post 12-week Intervention)
- Assessed changes in Fat-free Mass (percentage of Fat-free Mass)(Pre and Post 12-week Intervention)
- Assessed Height(Pre and Post 12-week Intervention)
- Assessed changes in Fat-free Mass (kilograms of Fat-free Mass)(Pre and Post 12-week Intervention)
- Assessed changes in Fat Mass (percentage of Fat Mass)(Pre and Post 12-week Intervention)
- Assessed changes in Fat Mass (kilograms of Fat Mass)(Pre and Post 12-week Intervention)
- Assessed changes in Body Mass Index(Pre and Post 12-week Intervention)
- Assessed changes in Body Weight(Pre and Post the 12-week intervention)
- Assessed changes in dietary intake: Frequency of consumption(Pre and post the 12-week intervention)
- Assessed changes in dietary intakes: 24 hours dietary recalls(Pre and post the 12-week intervention)
- Assessed changes in physical activity and sedentary time(Pre and post the 12-week intervention)
- Assessed changes in Maximal Fat Oxidation(Pre and post the 12-week intervention)
- Assessed changes in Relative Intensity of Maximal fat Oxidation during exercise(Pre and post the 12-week intervention)
- Assessed changes in Cardiorespiratory Fitness(Pre and post the 12-week intervention)