Efficacy of Educational Nutrition and Exercise on the Regulation of Appetite Through Exosomes in Type 2 Diabetics

University of Cadiz146 enrolled

Overview

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.

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.

Interventions

Nutritional InterventionBEHAVIORAL

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.

High-intensity interval training (HIIT)OTHER

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.

Moderate-intensity continuous training (MICT)OTHER

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.

Eligibility

Sex: ALLMin age: 30 YearsMax age: 65 Years

Medical Language ↔ Plain English

Inclusion Criteria: * 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 Exclusion Criteria: * 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

Outcomes

Primary Outcomes

Glycated hemoglobin

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

Time frame: Pre and Post the 12-week Intervention

Secondary Outcomes

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).

Time frame: Pre and Post the 12-week intervention

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).

Time frame: Pre and Post the 12-week intervention

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.

Time frame: Pre and Post the 12-week intervention

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.

Time frame: Pre and Post the 12-week intervention

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%.

Time frame: Pre and Post the 12-week intervention

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.

Time frame: Pre and Post the 12-week intervention

Assessed changes in Insulin Sensitivity: Quicki

Insulin sensitivity was estimated through the Quantitative Insulin Sensitivity Check Index (QUICKI), calculated as 1 / \[log(fasting insulin (µU/mL)) + log(fasting glucose (mg/dL))\].

Time frame: Pre and Post 12-week intervention

Assessed changes in Insulin Sensitivity: Matsuda Index

Whole body insulin sensitivity was estimated through the Matsuda Index, calculated as 10,000 / √\[(fasting glucose (mg/dL) × fasting insulin (µU/mL)) × (mean glucose during Oral Glucose Tolerance Test (mg/dL) × mean insulin during Oral Glucose Tolerance Test (µU/mL))\].

Time frame: Pre and Post 12-week intervention

Assessed changes in Insulin Resistance: HOMA-IR

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.

Time frame: Pre and Post 12-week intervention

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.

Time frame: Pre and Post 12-week Intervention

Assessed changes in Anthropometry: Waist to Hip Ratio

Waist-to-hip ratio (WHR) was calculated as waist circumference (cm) divided by hip circumference (cm).

Time frame: Pre and Post 12-week Intervention

Assessed changes in Anthropometry: Perimeters

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.

Time frame: Pre and Post 12-week Intervention

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.

Time frame: Pre and Post 12-week Intervention

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.

Time frame: Pre and Post 12-week Intervention

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.

Time frame: Pre and Post 12-week Intervention

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.

Time frame: Pre and Post 12-week Intervention

Assessed changes in Body Mass Index

Body Mass Index (BMI) in kilograms per square meter (kg/m²) was calculated as body weight (kg) divided by height squared (m²).

Time frame: Pre and Post 12-week Intervention

Assessed Height

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.

Time frame: Pre and Post 12-week Intervention

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.

Time frame: Pre and Post the 12-week intervention

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").