Diurnal Variation of Exercise on Metabolic Health

Study Overview
=================
Brief Summary
-----------------
The main objective of this project is to study the diurnal variation of the effect of exercise on glycemic metabolism and fat oxidation in humans.

Detailed Description
-----------------
Strong scientific evidence supports the beneficial effects of exercise on cardiovascular health, the regulation of glucose metabolism, and fat oxidation. Physical performance capacity is known to fluctuate throughout the day, however, it is unknown whether there is an optimal time of day to maximize the effects of exercise on health, and specifically on blood glucose metabolism and fat oxidation. Finding the ideal time to perform physical exercise is of clinical and public health interest. Likewise, optimizing the timing of physical exercise to coincide with the greater physiological response of each individual would mean increasing the potential of exercise as a therapeutic tool. Specific aims of this project are a) to describe possible differences dependent on sex and weight in the diurnal variation of the effect of exercise on glycemic metabolism and fat oxidation, and b) to characterize the molecular mechanisms implicated. 40 young adults with normal weight (50% females), and 40 young adults with obesity (50% females) will be randomized into two conditions (morning and evening) with at least 3 days of separation in between. Each evaluation will conform the following tests: Basal metabolic rate assessment through indirect calorimetry. Fuel oxidation and energy expenditure assesment during a 60 minutes bout of aerobic steady-state exercise (cycling), through indirect calorimetry. Fuel oxidation and energy expenditure assesment after exercise during 60 minutes, through indirect calorimetry. Blood samples assesment before, inmediately after, and 60 minutes after exercise. Quadriceps biopsies before, inmediately after, and 60 minutes after exercise. *This will be done only in a sub-cohort of 32 participants. Visual analog scales to assess appetite before, inmediately after, and 60 minutes after exercise. Continous glucose monitoring from the first session day until14 days past. Previous to this, participants' body composition and fitness level will be assesed via densitometry and a maximal exercise test, respectively.

Official Title
-----------------
Diurnal Variation of the Effect of Aerobic Exercise on Glycemic Metabolism and Fat Oxidation in Humans: Role of Sex and Body Weight

Conditions
-----------------
Aerobic Exercise, Acute Exercise, Circadian Rhythm Sleep-Wake Disorders, Unspecified Type, Glucose Metabolism, Lipid Metabolism, Diurnal Variation, Young Adults

Intervention / Treatment
-----------------
* Other: Exercise


Participation Criteria
=================
Eligibility Criteria
-----------------
Inclusion Criteria: Body mass index: 18,5-24,9 kg/m2 (normal weight group) and 30-39.9 (obesity group) kg/m2 Sedentary: physical activty <20 min, 3 days/week. To be able to understand instrucctions, objectives and study protocol. Exclusion Criteria: History of a major adverse cardiovascular event, kidney failure, cirrhosis, eating disorder, weight control surgery, or HIV / AIDS. Rheumatoid arthritis, Parkinson's disease, active cancer treatment in the past year, type 1 diabetes mellitus, or another medical condition for which fasting is contraindicated. Any condition that, in the judgment of the investigator, impairs the ability to participate in the study or poses a personal risk to the participant. Use of medications that may affect the results of the study. Unstable body weight for 3 months before the start of the study (> 4 kg weight loss or gain) Pregnancy and breastfeeding. Active tobacco abuse or illicit drug use or a history of alcohol abuse treatment. On a special diet or prescribed for other reasons (eg celiac disease).

Ages Eligible for Study
-----------------
Minimum Age: 20 Years
Maximum Age: 50 Years

Sexes Eligible for Study
-----------------
All

Accepts Healthy Volunteers
-----------------
Accepts Healthy Volunteers

Study Plan
=================
How is the study designed?
-----------------

Design Details

Primary Purpose: Prevention
Allocation: Randomized
Intervention Model: Crossover Assignment
Masking: Single


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Morning<br>60 minutes cycling on cycle ergometer in the morning (9:00). Steady-state test at an intensity of 10% less of participant's ventilatory threshold. | Other: Exercise<br>* Aerobic exercise<br>|
| Experimental: Evening<br>60 minutes cycling on cycle ergometer in the evening (18:00). Steady-state test at an intensity of 10% less of participant's ventilatory threshold. | Other: Exercise<br>* Aerobic exercise<br>|

What is the study measuring?
-----------------
Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change from resting blood glucose to 48 hours after exercise | A last generation continuous glucose monitor (DEXCOM G6) inserted into the skin of the abdominal area will be used to record and store blood glucose levels every 5 minutes, for 24 hours, 14 consecutive days. Within this time range both exercise conditions will be performed. The monitor will be placed 24 hours before the first exercise session and will be removed 48 hours after the second exercise session. Therefore, change from resting blood glucose 24 hours previous to 48 hours after exercise will be obtained for each condition. | 14 days | 
| Change from resting markers of glucose metabolism to 60 minutes after exercise | Blood samples will be taken after exercise, immediately after exercise and 60 minutes after exercise, in order to measure change in plasma concentration of markers of glucose metabolism from resting to 60 minutes after exercise. | 2 hours | 
| Change from resting fat oxidation to 60 minutes after exercise | Metabolic expenditure and fat oxidation will be measured at rest 30 minutes before exercise, during exercise, as well as after exercise during 60 minutes by indirect calorimetry (Vyntus CPX (Jaeger-CareFusion, Höchberg, Germany). | 3 hours | 
| Change from resting markers of fatty acid metabolism to 60 minutes after exercise | Blood samples will be taken to measure plasma concentration of markers of fatty acid metabolism before, immediately after and 60 minutes after exercise. In addition, 3 muscle biopsies will be performed in the vastus lateralis of the quadriceps, before exercise, immediately after exercise and 60 minutes after exercise. The expression of proteins related to the metabolism of fat oxidation and messenger RNA will be analyzed. In addition, a part of fresh tissue will be used to perform mitochondrial respiration assessments. | 2 hours | 
| Gene expression | Biopsies will be obtained by using microbiopsy needles (Achieve Automatic Needle 16G x 15 cm), with coaxial, obtaining a maximum of 30 mg per biopsy after previous local anaesthesia with 2% lidocaine. These biopsies will be performed by a surgeon with experience in this type of procedure who has previously collaborated with us. The distal part of the vastus lateralis of the quadriceps muscle will be obtained. The collected sample will be cleansed of visible blood and fat and split into several portions. A two-step qRT-PCR platform (Real Time ready Custom Panel 96, Roche, Barcelona, Spain) will be used. The RNA will be extracted with the PeqGOLD HP Total RNA kit (Peqlab, Germany), according to the manufacturer's recommendations. cDNA will be synthesized in a standard manner. The specific primers will be: PGC-1, AMPK, HIF1, HIF2, DRP and OPA1. | 2 hours | 
| Protein expression | To learn about the specific signalling, a portion of muscle (  15mg) will be used for protein extraction and quantification. The samples will be mixed with 3X SDS-PAGE, separated via SDS-PAGE and transferred to a nitrocellulose membrane. After incubation, the membranes will be loaded with the following antibodies: AMPK, AMPK p, Thr172, OPA1, OPA1 p, DRP, DRPp, CS, β-HAD, EL, LPL, FABPpm, CD36, Perilipines 2, 3 and 5, DGAT 1 and 2, ATGL, HSL, BMAL1, CLOCK, CRY 1 and 2, NR1D1 and ALAS 1. | 2 hours | 
| Mitochondrial breathing | To isolate fresh mitochondria, the fresh muscle portion will immediately be placed on ice. The final crude mitochondrial pellet will be re-suspended in 1X MAS medium. Mitochondrial respiration will be measured using an XFe24 extracellular flux analyzer (Seahorse Bioscience) sequentially in a coupled state with the different substrates (e.g. succinate, malate, glutamate and pyruvate) corresponding to complex 2 (basal respiration), followed by complex 3 (phosphorylative respiration in the presence of ADP and substrates). Complex 4 (non-phosphorylative or resting respiration) will be measured after the addition of oligomycin once all the ADP is consumed and, subsequently, the maximum respiration stimulated by an uncoupler (Complex 3u) will be measured. | 2 hours | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change from resting appetite to 60 minutes after exercise | Measures of appetite before, immediately after and 60 minutes after exercise using an appetite visual analog scale (appetite VAS) scoring from 0 to 10, where 0 is the minimum punctuation for appetite and 10 is the maximum punctuation for appetite. | 2 hours | 

 Terms related to the study
=================
Keywords Provided by Centre Hospitalier Valida
-----------------
Aerobic Exercise, Diurnal Variation, Circadian Rhythm, Glucose Metabolism, Lipid Metabolism, Young adults