Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Strain selection
• Procedure: Exercise programs
• Procedure: Body composition
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Weigh mice before and after treatment	Scales	Body weight
2	Assess body composition before and after treatment	MRI	Lean mass and fat mass
3	Collect metabolic measurements and measure food and water intake before and after treatment	Indirect calorimetry	See McMullan7

Equipment and supplies

• Treadmill (Exer 3/6, Columbus Instruments, Columbus OH)
• Whole body MRI (EchoMRI 3-in-1 Body Composition Analyzer, EchoMRI, Houston TX)

Reagents and solutions

• Disinfectant

Procedure: Strain selection

1. Four strains were selected based on the following criteria: 1) ability to run on treadmills, 2) fat response type to voluntary exercise, 3) endurance ability (MPD datasets: McMullan1, McMullan2, McMullan3, McMullan4; all data was collected in older females).
2. Selected strains were: CC002/Unc adverse fat responder, low endurance; CC027/GeniUnc adverse fat responder, high endurance; CC013/GeniUnc standard fat responder, low endurance; and CC037/TauUnc standard fat responder, high endurance.

Procedure: Exercise programs

1. High intensity interval training (HIIT): Five intervals with 80% max speed for four minutes, 20% max speed for one min, and ten 30 s transitions to decrease and increase speed between the different intensities.
2. Moderate intensity continuous training (MICT): Distance matched to the HIIT protocol and consists of ~43 min duration at 50% max speed.
3. No exercise (NE).
4. Mice complete 5 wks of exercise training on treadmills. Mice are acclimated to the treadmills for 3 days during the first week.
5. After acclimation, HIIT and MICT mice complete 4 wks of training, three times a week (all training occurs in the morning).
6. Compliance is tracked and mice with 50% or more non-compliant days are removed from statistical analysis. A mouse is considered non-compliant if it refuses or is not able to continue regular treadmill running despite extra stimulus from shock and/or prodding.

Procedure: Body composition

1. Body composition is assessed during mornings using whole body MRI to determine fat and lean mass content.
2. Measurements are taken prior to the start of the experiment and upon completion of exercise training.
3. Body fat and lean mass percentages were calculated relative to body mass at each time point in every experiment. Body mass response was calculated as [(Post-mass – pre-mass)/pre-mass] x 100. Body composition percentage response was calculated as [(post-measurement % - pre-measurement %)/pre-measurement %] x 100. Body mass and composition responses were calculated for the five weeks of exercise program treatment. Negative values represent a loss and positive values represent a gain in response to treatment.

Data collected by investigator

• Body weight and body weight response
  
• Body composition
  • Fat percentage and response
  • Lean percentage and response