Mitochondrial Metabolism and Oxidative Stress in Function of the Physical Exercise and Nutritional Counseling in T2DM

NCT07251504 | Active Not Recruiting

• 2 other identifiers: MITOXPP11-007-2023
• Study Type: interventional
• Target: 146
• Locations: 1 country
• Sites: 1

Brief Summary

The goal of this randomized controlled trial is to learn about the effects of different aerobic modalities (moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT)) and diet on mitochondrial properties and oxidative stress in patients with type 2 diabetes (T2D). It will also learn about the different effects of these interventions in men and women. The main questions it aims to answer are:

• What is the isolated and combined effect of these exercise modalities and diet? Is there a superior approach for mitochondrial metabolism and oxidative stress in patients with T2D?
• Is there a sex-specific best combination choice for these different interventions?
• Is there any relation between exercise and diet-induced changes in mitochondrial properties and oxidative stress and other health-related outcomes such as body composition or insulin sensitivity? To answer these questions, researchers designed a two-factor study. One factor was exercise, which had three levels (MICT, HIIT, and the inactive (INACT) condition). The second factor was diet, with two levels (Diet (D) and no diet (ND)). Hence, participants were randomly allocated into 6 groups: INACT-ND, INACT-D, MICT-ND, MICT-D, HIIT-ND, and HIIT-D. Participants did:
• Underwent a 12-week intervention within the condition of his/her group
• Visit the research group facilities three times per week if in an exercise group or once every two weeks if in a diet group.

Conditions

Diabetes Mellitus, Type 2

Keywords

metabolic disorders; aerobic exercise; muscle metabolism; high-resolution respirometry

Outcome Measures

Primary Outcomes (5)

• Muscle biopsy samples: mitochondrial respiratory capacity

  Skin was locally anesthetized, followed by an incision of approximately 6-7mm on the dermal surface. Using sterilized Bergstrom needles, approximately 3-6 mg of skeletal muscle from the lateral vastus were extracted and placed in a BIOPS buffer for immediate analysis of mitochondrial respiration (MR) through high-resolution respirometry. Before, the fibers were mechanically separated in BIOPS buffer using sterilized needles and chemically permeabilized with a 50 μg/ml saponin solution. MR was measured in a specific buffer (Mir05) in duplicate in hyperoxygenated chambers (250-450 mM of oxygen) in an Oxygraph-2k at 37ºC (Oroboros, Innsbruck, Austria). A substrate-uncoupler titration protocol was employed by adding (final concentration): malate (2mM), pyruvate (5mM), glutamate (2mM), MgCl2 (3mM), and ADP (5mM), cytochrome C (10μM), succinate (10mM), and p-trifluoromethoxyphenylhydrazone. Oxygen flux was presented as mass-specific (pmol·s/mg wet weight) and normalized to CS activity.

  Pre and post the 12-week intervention

• Assessed changes in Oxidative Stress

  The level of oxidative stress was assessed by the concentration of 8-OHdG as a biomarker of DNA oxidation. A competitive ELISA detection method was employed in serum samples. Briefly, the standard and the sample were added to a pre-coated microplate along with the biotinylated detection antibody. After washing, HRP-Streptavidin was added. Then, the TMB substrate was added, and the reaction was terminated with an acid solution. The color production was measured spectrophotometrically at a wavelength of 450nm. The optical density values of samples were compared with those of the standard curve to determine the 8-OHdG concentration (ng/ml).

  Pre and post the 12-week intervention

• Muscle biopsy sample: Citrate Synthase activity

  2-5 mg of frozen muscle was homogenized in a 1:20 dilution of CelLytic™ MT Tissue Lysis (C3228, Sigma-Aldrich, Dorset, UK), with 1 % protease inhibitor cocktail (P8340, Sigma-Aldrich, Dorset, UK) at 4 °C. The lysate was centrifuged at 12,000 × g for 10 min at 4 °C to pellet debris, and the supernatant was stored at -80 °C. CS activity was quantified in duplicates using an assay kit following the manufacturer's instructions (CS0720: Sigma-Aldrich, Dorset, UK). Briefly, endogenous activity was recorded through color emission by DTNB, and total activity was calculated after the addition of oxalacetate. Protein concentration was determined in duplicates using a bicinchoninic acid assay kit (BCA1: Sigma-Aldrich, Dorset, UK), enabling normalization of CS activity values to total protein content (µmol/g/min). CS activity and protein concentration were measured in 96-well plates using a microplate reader at 412 nm and 562 nm, respectively (MQX200R2, Biotek instruments, Burlington, USA).

  Pre and post the 12-week intervention

• Assessed changes in haemoglobin saturation delta

  A NIRO-200NX device was used to measure skeletal muscle changes on oxygenated, desoxygenated, and total haemoglobin (μmol/L) through near-infrared spectroscopy (NIRS) and the modified Beer-Lambert law. The NIRS sensor (emitter-sensor distance of 4cm) was placed at 2/3 of the distance between the major trochanter and the lateral epicondyle of the dominant leg. For parameters determined by MBLL, the reference point was set after five minutes of resting in a supine position. The NIRS data were continuously acquired throughout a resting energy expenditure test for 30 minutes, a maximal fat oxidation (MFO) test on a cycloergometer, and a maximal oxygen consumption (VO2max) test on a cycloergometer. Briefly, the MFO test consisted of an incremental task with 3-minute phases starting from 15W and increasing by 15W each phase until a respiratory exchange ratio of 1. After a 5-minute rest, the VO2max test was initiated at last MFO load with 15W increases each minute until VO2max determination.

  Pre and post the 12-week intervention

• Assessed changes in Tissue Oxygenation Index

  A NIRO-200NX device was used to measure skeletal muscle tissue oxygenation index (%)through near-infrared spectroscopy (NIRS), the modified Beer-Lambert law (MBLL), and spatially resolved spectroscopy. The NIRS sensor (emitter-sensor distance of 4cm) was placed at 2/3 of the distance between the major trochanter and the lateral epicondyle of the dominant leg. For parameters determined by MBLL, the reference point was set after five minutes of resting in a supine position. The NIRS data were continuously acquired throughout a resting energy expenditure test for 30 minutes, a maximal fat oxidation (MFO) test on a cycloergometer, and a maximal oxygen consumption (VO2max) test on a cycloergometer. Briefly, the MFO test consisted of an incremental task with 3-minute phases starting from 15W and increasing by 15W each phase until a respiratory exchange ratio of 1. After a 5-minute rest, the VO2max test was initiated at last MFO load with 15W increases each minute until VO2max determination.

  Pre and post the 12-week intervention

Secondary Outcomes (16)

• Assessed changes in Body composition: Fat mass and Fat-free mass.

  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 Insulin Resistance: HOMA-IR

  Pre and Post 12-week intervention

• Assessed changes in Insulin Sensitivity: Matsuda Index

  Pre and Post 12-week intervention

Study Arms (6)

ND-INACT

NO INTERVENTION

Participants who did not receive either nutritional intervention or exercise program. They were instructed to maintain their normal life habits regarding physical activity and diet.

Moderate-intensity continuous training (ND-MICT)

ACTIVE COMPARATOR

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

Other: The Moderate-Intensity Continuous Training (MICT)

High-intensity interval training (ND-HIIT)

ACTIVE COMPARATOR

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

Other: The High-Intensity Interval Training (HIIT)

Nutritional intervention (D-INACT)

ACTIVE COMPARATOR

Participants who received nutritional intervention but not an exercise program.

Behavioral: Nutritional intervention

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

EXPERIMENTAL

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

Behavioral: Nutritional intervention; Other: The Moderate-Intensity Continuous Training (MICT)

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

EXPERIMENTAL

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

Behavioral: Nutritional intervention; Other: The 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 over an entire period of 12 weeks. 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 High-intensity interval training (D-HIIT); Nutritional Intervention Moderate-Intensity continuous training (D-MICT); Nutritional intervention (D-INACT)

The 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 from a previously performed 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 40% 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)

The 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. MICT consisted of 50 minutes of continuous pedaling at an intensity approximately 10 % above the lactate threshold, with a cadence between 60-80 rpm. 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 previously performed FatOx test data, with 10% increments in each step. TL was determined by the phase in which HR increased by ≥ 3 beats between phases and blood lactate levels increased by ≥ 1 mmol/L from the previous phase.

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 (glycated hemoglobin ≥ 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
• Instituto de investigación e innovación biomédica de Cádiz: collaborator

Study Sites (1)

Science of Education Faculty

Puerto Real, Cádiz, 11510, Spain

Location

MeSH Terms

Conditions

Diabetes Mellitus, Type 2; Metabolic Diseases

Condition Hierarchy (Ancestors)

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

Study Officials

• Cristina Casals Vázquez, PhD

  University of Cádiz

  PRINCIPAL INVESTIGATOR

• Miguel Efrén Jiménez Romero, MD

  Hospital de la Línea de la Concepción

  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 trial in individuals with type 2 diabetes and obesity

Study Record Dates

• First Submitted: September 26, 2025
• First Posted: November 26, 2025
• Study Start: March 10, 2022
• Primary Completion: December 31, 2025
• Study Completion: December 31, 2025
• Last Updated: November 26, 2025

Record last verified: 2025-11

Data Sharing

• IPD Sharing: Will not share

Locations

ES (1)