教育性营养和锻炼对通过外泌体调节 2 型糖尿病患者食欲的疗效 (APETEX)

有源比较器：中等强度持续训练 (ND-MICT)

未接受营养干预但参加了中等强度连续训练运动项目的参与者。

其他：Moderate-intensity continuous training (MICT)

Participants assigned to the MICT completed 3 weekly sessions, with 1-2 days of rest between sessions, on a cycle ergometer for 12 weeks. The MICT program consisted of 50 minutes of continuous pedaling at an intensity approximately 10% above the lactate threshold, with cadence maintained between 60-80 rpm. The training load (TL) was increased by 10% if these two conditions were met: 1) no increase of at least 2 beats of heart rate from minute 20 to minute 40 of the training session, and 2) lactate levels below 2 mmol/L in minute 48 of the session. The TL for the first session was determined by an adjustment test consisting of an incremental test with five consecutive 10-minute phases at a constant cadence (60-80 RPM) from 90% to 130% of the ventilatory threshold power, calculated from the FatOx test data, with 10% increments in each step. The TL was determined by the phase in which HR increased by ≥ 3 beats between phases and blood lactate levels increased by ≥ 1 mmol/L between phases.

有源比较器：高强度间歇训练（ND-HIIT）

未接受营养干预但参加了高强度间歇训练运动项目的参与者。

其他：High-intensity interval training (HIIT)

Participants assigned to the HIIT completed 3 weekly sessions, with 1-2 days of rest between sessions, on a cycle ergometer for 12 weeks. The training load (TL) was determined from an incremental test until volitional exhaustion with one-minute work intervals (from 85% to 165% of maximal power output during VO2max test [Wmax] and with 10% increments) separated by 1.5-minute rest periods (at 20% Wmax). The program consisted of a 3 minutes warm-up with the first 2 minutes at 20% and last minute at 40% of the TL, followed by 10 series of 1 min duration at 90% of TL, with 1 minute of rest between sets at 20% of TL and ending with 2 minutes of cool-down at 20% of TL (estimated total time of the session: 25 minutes). A 5% increase in workload was applied when, for two consecutive sessions, the first three intervals failed to exceed 85% of maximal HR and at least five did not reach 90% of maximal HR. Participants were asked to maintain a cadence above 80RPM.

实验性的：营养干预高强度间歇训练（D-HIIT）

接受了营养干预并参加了高强度间歇训练运动计划的参与者。

行为的：Nutritional Intervention

The nutritional intervention was conducted through biweekly (± 3 days) in-person consultations with a nutritionist or dietitian. Firstly, the patient's diet was analyzed. Then, participants were guided to follow a mildly calorie-restricted diet (-250-500 kcal/day deficit), with the primary goal of achieving a 5 % reduction in body weight and reducing blood glucose levels. Meal plans were created using Nutrium® software , personalized to each participant's lifestyle and food preferences, and reviewed at each biweekly consultation. The dietary plan included at least 4 or 5 meals per day, ensuring a well-distributed intake of carbohydrates, a balanced distribution of macro- and micronutrients, and the inclusion of foods rich in prebiotics and probiotics. Moreover, some suggestions about the combination of foods and culinary techniques were provided. Only one session through the intervention was allowed to be attended telephonically.

其他：High-intensity interval training (HIIT)

Participants assigned to the HIIT completed 3 weekly sessions, with 1-2 days of rest between sessions, on a cycle ergometer for 12 weeks. The training load (TL) was determined from an incremental test until volitional exhaustion with one-minute work intervals (from 85% to 165% of maximal power output during VO2max test [Wmax] and with 10% increments) separated by 1.5-minute rest periods (at 20% Wmax). The program consisted of a 3 minutes warm-up with the first 2 minutes at 20% and last minute at 40% of the TL, followed by 10 series of 1 min duration at 90% of TL, with 1 minute of rest between sets at 20% of TL and ending with 2 minutes of cool-down at 20% of TL (estimated total time of the session: 25 minutes). A 5% increase in workload was applied when, for two consecutive sessions, the first three intervals failed to exceed 85% of maximal HR and at least five did not reach 90% of maximal HR. Participants were asked to maintain a cadence above 80RPM.

无干预：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.

有源比较器：Nutritional Intervention (D-INACT)

Participants who received nutritional intervention but not an exercise program.

行为的：Nutritional Intervention

The nutritional intervention was conducted through biweekly (± 3 days) in-person consultations with a nutritionist or dietitian. Firstly, the patient's diet was analyzed. Then, participants were guided to follow a mildly calorie-restricted diet (-250-500 kcal/day deficit), with the primary goal of achieving a 5 % reduction in body weight and reducing blood glucose levels. Meal plans were created using Nutrium® software , personalized to each participant's lifestyle and food preferences, and reviewed at each biweekly consultation. The dietary plan included at least 4 or 5 meals per day, ensuring a well-distributed intake of carbohydrates, a balanced distribution of macro- and micronutrients, and the inclusion of foods rich in prebiotics and probiotics. Moreover, some suggestions about the combination of foods and culinary techniques were provided. Only one session through the intervention was allowed to be attended telephonically.

实验性的：Nutritional Intervention Moderate-intensity continuous training (D-MICT)

Participants who received nutritional intervention and were enrolled in a moderate-intensity continuous training exercise program.

行为的：Nutritional Intervention

The nutritional intervention was conducted through biweekly (± 3 days) in-person consultations with a nutritionist or dietitian. Firstly, the patient's diet was analyzed. Then, participants were guided to follow a mildly calorie-restricted diet (-250-500 kcal/day deficit), with the primary goal of achieving a 5 % reduction in body weight and reducing blood glucose levels. Meal plans were created using Nutrium® software , personalized to each participant's lifestyle and food preferences, and reviewed at each biweekly consultation. The dietary plan included at least 4 or 5 meals per day, ensuring a well-distributed intake of carbohydrates, a balanced distribution of macro- and micronutrients, and the inclusion of foods rich in prebiotics and probiotics. Moreover, some suggestions about the combination of foods and culinary techniques were provided. Only one session through the intervention was allowed to be attended telephonically.

其他：Moderate-intensity continuous training (MICT)

Participants assigned to the MICT completed 3 weekly sessions, with 1-2 days of rest between sessions, on a cycle ergometer for 12 weeks. The MICT program consisted of 50 minutes of continuous pedaling at an intensity approximately 10% above the lactate threshold, with cadence maintained between 60-80 rpm. The training load (TL) was increased by 10% if these two conditions were met: 1) no increase of at least 2 beats of heart rate from minute 20 to minute 40 of the training session, and 2) lactate levels below 2 mmol/L in minute 48 of the session. The TL for the first session was determined by an adjustment test consisting of an incremental test with five consecutive 10-minute phases at a constant cadence (60-80 RPM) from 90% to 130% of the ventilatory threshold power, calculated from the FatOx test data, with 10% increments in each step. The TL was determined by the phase in which HR increased by ≥ 3 beats between phases and blood lactate levels increased by ≥ 1 mmol/L between phases.

Glycated hemoglobin

Glycated hemoglobin (HbA1c, %) was measured using high-performance liquid chromatography (HPLC) with the automated ADAMS™ HA-8180V analyzer (ARKRAY, Kyoto, Japan).

评估胰岛素抵抗的变化：HOMA-IR

胰岛素抵抗通过稳态模型评估胰岛素抵抗（HOMA-IR）进行估算，计算公式为空腹胰岛素（μU/mL）乘以空腹血糖（mg/dL）再除以405。

评估胰岛素敏感性变化：松田指数

全身胰岛素敏感性通过松田指数估算，计算公式为：10,000 / √[(空腹血糖 (mg/dL) × 空腹胰岛素 (µU/mL)) × (口服葡萄糖耐量试验期间平均血糖 (mg/dL) × 口服葡萄糖耐量试验期间平均胰岛素 (µU/mL))]。

评估胰岛素敏感性变化：Quicki

胰岛素敏感性通过定量胰岛素敏感性检测指数（QUICKI）进行评估，计算方式为 1 / [log(空腹胰岛素（微单位/毫升）) + log(空腹血糖（毫克/分升）)]。

评估了体重指数的变化

体重指数（BMI）以千克每平方米（kg/m²）为单位，计算公式为体重（千克）除以身高的平方（平方米）。

评估人体测量学变化：周长

腰围（厘米）在第十肋骨下外侧缘与髂嵴之间的中点处测量，垂直于躯干纵轴。 腹围（厘米）在脐部水平处测量，垂直于躯干纵轴。 臀围（厘米）在臀部最突出处水平测量，垂直于躯干纵轴。 大腿围（厘米）在股骨大转子与胫骨外侧髁之间的中点处测量。 所有测量均在参与者直立、放松状态下进行。 每项测量进行两次，如果差异超过1%，则进行第三次测量。 记录两次最相似测量值的平均值。

评估人体测量学变化：腰臀比

腰臀比（WHR）的计算方式为腰围（cm）除以臀围（cm）。

评估最大脂肪氧化的变化

最大脂肪氧化通过自行车测功计（Lode Excalibur，荷兰格罗宁根）上的渐进测试确定。 气体交换（VO2、VCO2、RER）使用间接热量测定法（Jaeger MasterScreen CPX®）记录，心率通过Polar Team 2（Polar Electro Inc.，纽约州成功湖）在测试过程中连续测量。 测试包括起始负荷15W，每3分钟增加15W，直到呼吸商达到稳定的值1。 在整个测试过程中，保持60转/分钟的节奏。 脂肪氧化（克/分钟）值根据每个3分钟阶段的最后一分钟平均的氧气和碳水化合物数据，使用Frayn化学计量方程估算，然后记录较高的脂肪氧化值。

评估运动期间最大脂肪氧化相对强度的变化

最大脂肪氧化相对强度（FatMax）反映了脂肪氧化达到峰值时的运动强度（以最大摄氧量（VO2max）百分比表示）。 FatMax通过分级最大脂肪氧化（MFO）自行车测试在自行车测功计（Lode Excalibur，荷兰格罗宁根）上测定。 测试从15W开始，每3分钟增加15W，直至呼吸商达到1，保持60rpm的转速。 通过间接测热法（Jaeger MasterScreen CPX®）测量气体交换（VO2、VCO2、RER），并持续监测心率（Polar Team 2，Polar Electro，纽约）。 MFO测试后，从MFO测试最后负荷开始进行第二次增量测试，以15W/分钟的增量直至力竭，以测定VO2max。 使用Frayn方程计算脂肪氧化速率（g/分钟），并绘制相对于VO2（%VO2max）的曲线。 与最大脂肪氧化对应的VO2被确定为FatMax。

评估心肺适能的变化

最大摄氧量（VO2max，ml/min）通过最大脂肪氧化（MFO）测试后，在自行车测功计（Lode Excalibur）上进行递增负荷测试进行评估。 气体交换（VO2、VCO2和RER）使用间接热量计（Jaeger MasterScreen CPX®）记录，心率通过Polar Team 2（Polar Electro Inc）在整个测试过程中持续测量。 在MFO测试后休息5分钟，VO2max阶段从MFO阶段达到的最后负荷开始，每分钟增加15瓦，保持70-90转/分钟的踏频，直至主观力竭。 当至少满足以下三个标准中的三个时，确认最大努力：VO2平台期（最后30秒内VO2-时间斜率<0.05 L·min-1）、达到年龄预测最大心率的≥90%、呼吸交换比（RER）≥1.10、Borg量表感知劳累度为8-10分，以及主观疲劳。 如果未达到三个标准，则记录的VO2值记为VO2peak。

评估身高

身高（米，m）使用壁挂式身高计（TANITA-LEICESTER HR-001，百利达公司，日本东京）测量，参与者根据制造商的说明站立。

Assessed changes in Body Weight

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 at least 48 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

Assessed changes in Fat Mass (kilograms of Fat Mass)

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 at least 48 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

Assessed changes in Fat Mass (percentage of Fat Mass)

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 at least 48 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)

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 at least 48 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)

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 at least 48 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

Assessed changes in Anthropometry: Skinfold thickness

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 1mm. The mean of the two most similar records was recorded.

Assessed changes in Glucose concentration

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, 150 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 at least 48 hours. Blood samples were centrifuged and plasma was analysed using the colorimetric method performed on the Alinity CI system (Abbott Laboratories, Abbott Park, IL, USA).

Assessed changes in Insulin concentration

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, 150 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 at least 48 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 Appetite assessment.

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) was completed to measure the participant's morning appetite. Likewise, they were 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 task of emotional reactivity to food images

Emotional reactivity to food images was 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 were 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 were 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 was conducted to assess baseline alpha activity (8-14 Hz). The experimental task presented 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 hormones

Appetite-regulating hormones were assessed from blood samples collected after an 8-10-hour overnight fast, with participants seated at rest for 15 minutes. Fasting samples were drawn from the antecubital vein using EDTA tubes for appetite hormones and CAT tubes for serum cortisol. EDTA tubes were 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 were 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 Plasma Exosomal miRNA expression

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 were 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.

Assessed changes in dietary intake: Frequency of consumption

One week before laboratory measurements, participants completed a 5-day weighed food record, including one weekend days, to capture habitual dietary intake. Additionally, dietary habits were assessed through a 137-item semi-quantitative Food Frequency Questionnaire (FFQ), previously validated in a Mediterranean Spanish population. The FFQ was administered in face-to-face interviews by trained nutritionists and included information on vitamin/mineral supplements and alcohol consumption. Participants reported their habitual intake over the previous year using a 9-level frequency scale (ranging from "never or almost never" to ">6 times/day").

Assessed changes in dietary intakes: 24 hours dietary recalls

Nutrient intakes were estimated using the DIAL software (version 3.15; Alce Ingeniería, Madrid, Spain), a validated tool for quantitative and qualitative assessment of dietary intake in the Spanish population. The software converted food consumption into daily energy and nutrient values, expressed absolute intake (g/day), percentage of Recommended Intake (RI) coverage, and the energy distribution profile (percentage of total energy from proteins, fats, carbohydrates, and alcohol).

Assessed changes in dietary intakes: Mediterranean Diet Adherence

Adherence to the Mediterranean Diet was evaluated using a validated 14-item questionnaire for the Spanish population, with scores ranging from 0 (lowest adherence) to 14 (highest adherence). The resulting score was classified into three categories: low adherence (range: 0-5), moderate adherence (range: 6-9), and high adherence to MedDiet (range: 10-14).

Assessed changes in accelerometry: Physical activity time

Physical activity and inactivity were assessed using triaxial accelerometers (GENEActiv; ActivInsights Ltd., Kimbolton, UK) placed on the non-dominant wrist from 5 to 7 consecutive days (direct method). Participants were instructed to wear the device at all times, including during sleep and water-based activities, to ensure comprehensive data capture. Valid data were defined as a minimum wear of 16 hours per day for at least four days (three weekdays and one weekend day) following established protocols (10.1177/07334648231218095). Accelerometers were set to 60 Hz (10.1111/sms.12795, 10.1249/MSS.0000000000000289). Raw data were downloaded using GENEActiv software version 3.3 and processed using the GGIR package (version 3.0.9) in R (v4.1.1, R Core Team, Vienna, Austria), which auto-calibrates the data based on local gravity and calculates the Euclidean Norm Minus One (ENMO) to minimize sensor calibration error (https://doi.org/10.1152/japplphysiol.00421.2014).

Assessed changes in physical activity and sedentary time

Physical activity, sedentary time, and sleep were also estimated through self-reported questionnaires, using the International Physical Activity Questionnaire Short Version (IPAQ-SF), previously validated in Spain. The IPAQ-SF provides estimates of sedentary time, moderate and vigorous activity in minutes per week.

Assessed changes in Resting Energy Expenditure

After at least 8 hours of fasting, resting energy expenditure was estimated by indirect calorimetry. 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 at least 48 hours. Participants lay supine for 30 minutes wearing a mask connected to a gas analyzer (open circuit, Jaeger MasterScreen CPX® (CareFusion, San Diego, USA)). The resting rates of fat and carbohydrate oxidation were used to determine the resting energy expenditure in kcal per minute (kcal/min).

Assessed changes in self-reported quality of life

Self-reported quality of life was registered by applying the Short Form 36-health survey (SF-36) questionnaire previously validated in Spain. The SF-36 is a generic measure of quality of life and has been evaluated for a wide variety of medical conditions, including diabetes. The SF-36 includes 36 questions that evaluate 8 subscales, scores for each scale range from 0 to 100, with higher scores indicating higher level of function or wellbeing. The physical component summary is derived from the 4 subscales of physical functioning, role physical, bodily pain, and general health; while the mental component summary is derived from the subscales of vitality, social functioning, role emotional, and mental health.

Assessed changes in blood pressure

Systolic and diastolic blood pressures were recorded three times by using the validated Omron HEM 742 blood pressure monitoring device with the participant seated, back supported in chair and feet flat on floor without legs crossed, after they rested 5 minutes. The guidelines of the Hypertension and Cardiology European Societies and the Spanish ones were used to classify blood pressure stages of participants.