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

NCT05259449 | Active Not Recruiting

• 2 other identifiers: The APETEX projectPID2020-120034RA-I00/AEI/10.13
• Study Type: interventional
• Target: 146
• Locations: 1 country
• Sites: 1

Brief Summary

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 type 2 diabetes (T2D) and 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, and D-HIIT. Participants will:
• Undergo a 12-week intervention within their assigned group.
• Visit the research facilities three times per week if assigned to an exercise group, or once every two weeks if in a diet group.

Conditions

Diabetes Mellitus, Type 2

Keywords

Insulin Sensitivity; Appetite regulation; aerobic exercise; Extracellular vesicles

Outcome Measures

Primary Outcomes (19)

• 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, 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).

  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, 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).

  Pre and Post the 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.

  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 / √\[(fasting glucose (mg/dL) × fasting insulin (µU/mL)) × (mean glucose during Oral Glucose Tolerance Test (mg/dL) × mean insulin during Oral Glucose Tolerance Test (µU/mL))\].

  Pre and Post 12-week intervention

• Assessed changes in Insulin Sensitivity: Quicki

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

  Pre and Post 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 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 expressions (Cq values) were normalized using reference miRNAs. Relative expression was determined with the 2\^-ΔCq method, and fold changes were calculated using the 2\^-ΔΔCq method, ensuring accurate quantification of miRNA expressions from exosomal RNA.

  Pre and Post the 12-week intervention

• Assessed changes in Appetite hormones

  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%.

  Pre and Post the 12-week intervention

• Assessed changes in task of emotional reactivity to food images

  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.

  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) 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.

  Pre and Post the 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 72 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

  Pre and Post the 12-week intervention

• Assessed in 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.

  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).

  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 72 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

  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 72 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

  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 72 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

  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 72 hours. Participants wore light clothing and were asked to urinate immediately before the measurement.

  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.

  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).

  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 5%. The mean of the two most similar records was recorded.

  Pre and Post 12-week Intervention

Secondary Outcomes (11)

• 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 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 physical activity and sedentary time

  Pre and post the 12-week intervention

Study Arms (6)

ND-INACT

NO INTERVENTION

Participants who do not receive either nutritional intervention or an exercise program. They will be instructed to maintain their normal life habits with respect to physical activity and diet.

Moderate-intensity continuous training (ND-MICT)

ACTIVE COMPARATOR

Participants who do not receive nutritional intervention but are enrolled in a moderate-intensity continuous training exercise program.

Other: Moderate-intensity continuous training (MICT)

High-intensity interval training (ND-HIIT)

ACTIVE COMPARATOR

Participants who do not receive nutritional intervention but are enrolled in a high-intensity interval training exercise program.

Other: High-intensity interval training (HIIT)

Nutritional Intervention (D-INACT)

ACTIVE COMPARATOR

Participants who receive nutritional intervention but not an exercise program.

Behavioral: Nutritional Intervention

Nutritional Intervention Moderate-intensity continuous training (D-MICT)

EXPERIMENTAL

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

Behavioral: Nutritional Intervention; Other: Moderate-intensity continuous training (MICT)

Nutritional Intervention High-intensity interval training (D-HIIT)

EXPERIMENTAL

Participants who receive nutritional intervention and are enrolled in a high-intensity interval training exercise program.

Behavioral: Nutritional Intervention; Other: High-intensity interval training (HIIT)

Interventions

Nutritional Intervention BEHAVIORAL

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 Mediterranean pattern-based and mildly calorie-restricted diet, 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 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.

Nutritional Intervention (D-INACT); Nutritional Intervention High-intensity interval training (D-HIIT); Nutritional Intervention Moderate-intensity continuous training (D-MICT)

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%, and the last five did not reach 90% of maximal HR. Participants were asked to maintain a cadence above 80RPM.

High-intensity interval training (ND-HIIT); Nutritional Intervention High-intensity interval training (D-HIIT)

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 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 training load 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. 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.

Moderate-intensity continuous training (ND-MICT); Nutritional Intervention Moderate-intensity continuous training (D-MICT)

Eligibility Criteria

• Age: 18 Years - 65 Years
• Sex: all
• Healthy Volunteers: No
• Age Groups: Adult (18-64), Older Adult (65+)

You may qualify if:

• Diagnosis of T2D or prediabetes (HbA1c ≥ 5.7%)
• No history of substance abuse (tobacco, alcohol, or other drugs).
• Body mass index \>25 kg/m², maintaining the habitual dietary patterns with stable body weight for at the least 6 months
• Stable physical activity and medication regimen for at least 6 months
• 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 tumours 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 a communication in Spanish or English.
• Not pregnant

You may not qualify if:

• Failure to attend more than 2 consecutive 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

Sponsors & Collaborators

• University of Cadiz: lead
• Ministerio de Ciencia e Innovación, Spain: collaborator

Study Sites (1)

Science of Education Faculty

Puerto Real, Cadiz, 11510, Spain

Location

MeSH Terms

Conditions

Diabetes Mellitus, Type 2; Insulin Resistance

Interventions

High-Intensity Interval Training

Condition Hierarchy (Ancestors)

Diabetes Mellitus; Glucose Metabolism Disorders; Metabolic Diseases; Nutritional and Metabolic Diseases; Endocrine System Diseases; Hyperinsulinism

Intervention Hierarchy (Ancestors)

Physical Conditioning, Human; Exercise; Motor Activity; Movement; Musculoskeletal Physiological Phenomena; Musculoskeletal and Neural Physiological Phenomena

Study Officials

• Cristina Casals, PhD

  University of Cadiz

  PRINCIPAL INVESTIGATOR

• Jesús Ponce González, PhD

  University of Cadiz

  PRINCIPAL INVESTIGATOR

Study Design

• Study Type: interventional
• Phase: not applicable
• Allocation: RANDOMIZED
• Masking: SINGLE
• Who Masked: OUTCOMES ASSESSOR
• Purpose: TREATMENT
• Intervention Model: PARALLEL
• Sponsor Type: OTHER
• Responsible Party: SPONSOR

Model Details: The project is a randomized controlled clinical trial in individuals with T2DM and obesity

Study Record Dates

• First Submitted: December 17, 2021
• First Posted: February 28, 2022
• Study Start: March 10, 2022
• Primary Completion: December 10, 2025
• Study Completion: December 10, 2025
• Last Updated: November 24, 2025

Record last verified: 2025-09

Data Sharing

• IPD Sharing: Will not share

Locations

ES (1)