Reprogramming Energy Homeostasis in Overweight Individuals Via Exercise, Cognitive, and Social Training

Leptin sensitivity will be evaluated as the ratio between caloric intake during an experimental meal and circulating leptin levels. Participants will receive a standardized breakfast and after 3 hours an ad libitum lunch, with detailed records of consumption, additional food requests, and portion sizes. Hunger and satiety will be measured using a 5-item Visual Analogue Scale (VAS) at multiple time points, recorded in millimeters. Energy and nutrient intake will be assessed using nutritional labels from each food item, and leptin sensitivity will be determined via plasma fasting.

Since direct assessment of leptin sensitivity in the central nervous system is not feasible in humans, peripheral leptin sensitivity ex vivo will be evaluated using Peripheral Blood Mononuclear Cells (PBMCs). PBMCs will be collected from the same individuals before and after the intervention and exposed to recombinant leptin to assess intracellular signalling activation. The primary leptin effector pathway is Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3), though Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activation has also been reported among others. The activation of these pathways will be measured using immunoassays such as Enzyme Linked Immunosorbent Assay (ELISA) and Western blot, quantifying the ratio of phosphorylated to total protein levels for each intracellular effector.

Insulin sensitivity and secretion will be assessed both during fasting and the oral glucose tolerance test (OGTT).

Energy metabolism, including resting metabolic rate and diet-induced thermogenesis, will be measured by indirect calorimetry.

Fat mass will be estimated using bioelectrical impedance analysis and expressed as a percentage of total body weight.

Fat mass will be estimated using bioelectrical impedance analysis and expressed in kilograms.

Assessed using the Colored Progressive Matrices (CPM), a standardized test that measures the ability to recognize patterns and solve problems without reliance on language. The outcome is the global Intelligent Quotient (IQ) score, with a mean of 100 and a standard deviation of 15, measured in standardized scores.

Evaluated through the N-Back Task, a computerized assessment of both visuo-spatial and verbal working memory. In the visuo-spatial condition, participants view randomly positioned blue boxes and determine whether the current position matches the previous one. In the verbal condition, they listen to a stream of letters and identify whether each letter matches the one immediately preceding it. The task adapts dynamically, increasing in difficulty when accuracy reaches or exceeds 80%. The outcome metric is the accuracy percentage at each difficulty level, measured in percentage of correct responses.

Measured using the Continuous Performance Test-II (CPT-II), a computerized assessment of sustained attention and inhibitory control. Participants respond to any letter except "X" (Go trials) and withhold responses when "X" appears (No-Go trials). Reaction times and response variability provide insights into attentional performance. The outcome metrics include accuracy, reaction time (RT) in milliseconds, and reaction time variability (RTV) in milliseconds.

To assess episodic long-term memory, the Verbal and Visual-Spatial Learning tasks from the standardized Battery for the evaluation of Memory (PROMEA) will be administered. The Verbal Learning Task presents a list of 15 unrelated words across 3 trials, followed by delayed recall after 15 minutes. Similarly, the Visual-Spatial Learning Task involves recalling the placement of 15 objects in 4 quadrants during 3 trials, with delayed recall after 15 minutes. The outcome metrics include the number of words or objects recalled across trials and the delayed recall score, expressed in standard scores and percentile ranks.

Assessed using the Test of Visual Motor Integration (VMI), which evaluates the coordination between visual perception and motor function. It consists of 3 components: Visual Motor Integration, requiring participants to copy increasingly complex forms; Visual Perception, involving the identification of matching forms among distractors; and Fine Motor Coordination, assessing precision in connecting dots within defined boundaries. The outcome includes raw scores converted to standard scores, with a mean of 100 and a standard deviation of 15, as well as percentile ranks.

Measured using the Movement Assessment Battery for Children-2 (MABC-2), which evaluates motor skills through 3 components: Manual Dexterity, assessing fine motor control; Aiming and Catching, measuring coordination between fine and gross motor movements; and Balance, which examines static and dynamic postural control through structured physical tasks. The outcomes are expressed as standard scores (mean = 100, SD = 15) and percentile ranks.

Evaluated using the Battery for Neuropsychological Evaluation of Children Battery (BVN 5-11), including the Digit Span and Corsi Block-Tapping Tests. The Digit Span Test requires participants to repeat sequences of numbers in the same or reversed order, assessing verbal short-term memory. The Corsi Block-Tapping Test involves replicating sequences of tapped blocks to assess visuo-spatial short-term memory. The score is determined by the number of items correctly recalled, expressed as Z-scores based on a normative sample, where a Z-score of 0 indicates the average performance of the normative group.

Assessed using the Child Behavior Checklist for Ages 6-18 (CBCL 6-18), a standardized parent-report questionnaire, comprised of 8 syndrome scales (anxious/depressed, withdrawn/depressed, somatic complaints, social problems, thought problems, attention problems, rule-breaking behaviour, and aggressive behaviour), 3 broadband scores (internalizing and externalizing problems, and total problem), Diagnostic and Statistical Manual of mental disorders (DSM)-oriented scales (affective problems, anxiety problems, somatic problems, attention deficit/hyperactivity problem, oppositional defiant problems, conduct problems), and the 2007 Scales (sluggish cognitive tempo, obsessive-compulsive problems, post-traumatic stress problems). For each subscale raw scores will be converted in T-scores (M = 50, SD = 10).

Ankle dorsiflexion will be evaluated using the Knee to Wall test, in which the participant places one foot touching the wall and gradually moves back until being able to touch the wall with the knee while keeping the heel on the ground.

Joint mobility will be assessed through a Scapular-Humeral Circumduction test, conducted with a calibrated stick, where the result is the last completed circumduction with extended arms.

Trunk strength will be assessed using the Trunk Lift test, in which participants lie prone on a mat and lift their upper body off the floor to their maximum height. The distance between the floor and the participant's chin will be measured.

In the Sit and Reach test, participants sit with their legs extended and feet dorsiflexed against a 30 cm support, then perform a forward trunk flexion. The result is measured as the distance between the fingertips and the toes.

Flexibility will also be assessed using the Lateral Trunk Flexion test, which records the difference between the initial hand position and the maximum lateral flexion.

The Pediatric Quality of Life Inventory (PedsQL version 4) is a widely used tool in studies involving children with obesity to measure key dimensions of health and quality of life. It includes domains of Physical, Emotional, Social, and School Functioning. Both child self-report and parent-report formats will be administered. Total Score and Psychosocial Health Summary Score will be calculated as percentages. Higher percentages indicate better functioning across these domains.

The social skill domain will be investigated through the standardized parent-reported Adaptive Behavior Assessment System, Second Edition (ABAS-II), which will provide the Social Adaptive Composite (SAC), which summarizes performance across the Leisure and Social skill areas. Raw scores will be converted into standardized weighted scores (M = 10, SD = 3) and standard scores (M = 100, SD = 15).

The Social Responsiveness Scale (SRS) will be used as well. The SRS is a standardized parent-report measure frequently used with children and adolescents between the ages of 4 and 18 years to screen subtle social communication deficits. The SRS generates 5 subscales: social awareness, social cognition, social motivation and social communication. Raw scores will be converted to gender-normed T-scores (M = 50, SD = 10).

The Eating Disorders Questionnaire in Childhood (EDQ-C) is a standardized tool used for the early identification of eating disorders in children, assessing comorbidities with attachment patterns, anxiety and mood disorders, as well as relational and behavioural difficulties. It includes a self-report version for children aged 8 and older, and 2 parent-report forms based on the child's age (4-7 and 8-12 years). For each diagnostic category, raw scores will be calculated and converted into standard score and percentile ranks (< 90 = within normal range, > 95 = clinically significant, > 99 = pathological).

To assess sleep habits and quality, the Sleep Disturbance Scale for Children (SDSC) will be employed. This standardized parent-report questionnaire evaluates various aspects of children's sleep, including duration, onset time, sleep disorders and an overall measure of sleep disturbances in children and adolescents aged 6 to 15 years. Six main areas are investigated by the following subscales: Disorders in Initiating and Maintaining Sleep (DIMS), Sleep Breathing Disorders (SBD), Arousal Disorders (DA), Sleep-Wake Transition Disorders (SWTD), Excessive Sleepiness (DES), and Nocturnal Hyperhidrosis (NH). For each subscale raw scores will be converted in T-scores (M = 50, SD = 10).

Brain responses will be studied through vascular brain activity to a glucose challenge, measured using functional near-infrared spectroscopy (fNIRS) in children and functional MRI (fMRI) in adults.

Plasma samples will be analysed for circulating levels of adipokines and cytokines (R&D Systems, Inc. 614 McKinley Place NE Minneapolis, MN 55413), including Brain-Derived Neurotrophic Factor (BDNF), a key regulator of neural plasticity and appetite suppression, Pro-OpioMelanoCortin (POMC) and its derivative Melanocyte-Stimulating Hormone (MSH), which play a central role in the anorectic response to leptin, eotaxin/CCL11, a proinflammatory chemokine linked to age-related cognitive decline, and Insulin-like Growth Factor-Binding Protein 2 (IGFBP2), a liver-derived protein regulated by leptin and involved in glucose homeostasis.

The composition of the gut microbiota will be assessed using genomic analysis of stool samples collected from participants. Fresh stool samples will be collected by participants at home, immediately frozen at approximately -20°C, and transported in a refrigerated box to the biobank. Upon arrival, the samples will be stored at -80°C until DNA extraction.

Upper limb strength will be evaluated with a Floor Press test using calibrated dumbbells based on each participant's strength.

Handgrip strength will be assessed using a dynamometer.

Lower limb explosive strength will be measured with the Standing Long Jump test, where participants jump as far as possible, landing with both feet together; the best of two attempts will be considered.

Abdominal strength and endurance will be assessed with a Sit-Up test, where participants lie supine with their legs bent at 90° and perform sit-ups, with the result being the maximum number of repetitions performed.

Balance will be assessed using the Single Leg Stance test, where participants maintain a single-leg stance within a shoe-sized rectangle for 60 seconds, with the number of falls recorded.