Procedures

General Information for All Procedures: Strains are entered into the phenotyping program randomly, and are entered into the program in staggered cohorts, typically of 2 weeks. All strains entered with both dietary cohorts at the same time, except strains BXD50, 68, 69, 71, 84, 85, 89, 95, 96, and 101; where CD cohorts entered before HFD cohorts. All cohorts consisted of littermates.

Procedure 1: Diet administration

Reagents and solutions

• Standard rodent chow (CD), 6% kCal fat, 20% kCal protein and 74% kCal of carbohydrates (Harlan, 2018, Indianapolis, IN, USA)
• High-fat diet (HFD), 60% kCal fat, 20% kCal protein and 20% kCal of carbohydrates (Harlan, 06414, Indianapolis, IN, USA)

Steps

1. All mice were fed a chow diet until 8 weeks of age.
2. At 8 weeks of age half the mice were fed a high-fat diet and the rest continued to be fed a chow diet.

Procedure 2: Body weight

Equipment, software, and supplies

• Balance scale

Steps

1. Body weights are recorded weekly from 8 to 29 weeks.
2. In addition to weekly recordings body weights are measured before each phenotypic test.

Procedure 3: Body composition analysis by qNMR

Definitions & Abbreviations: qNMR # Quantitative Nuclear Magnetic Resonance

Equipment, software, and supplies

• Body composition analyzer, whole body composition for live animals (Echo Medical Systems, 3-in-1 , Houston, TX, USA)

Steps

1. Whole body composition of fat and lean tissue mass is assessed by a qNMR. Evaluation is carried out before phenotypic tests at 16, 23 and 25 weeks of age.

Procedure 4: Food intake

Equipment, software, and supplies

• Individual metabolic cages, with food provided ad libitum in a feeder placed at the bottom of the cage. The cage does not contain any bedding nor enrichment/toys (Columbus Instruments, Comprehensive Lab Animal Monitoring System (CLAMS), Columbus, OH, USA)

Steps

1. Food was provided ad libitum during the study, however food intake is measured for individual animals while the mice are placed in the single animal metabolic cages at 16 and 23 weeks respectively. The amount of food available in the feeder is measured at the start and end of a 24h period in order to estimate the individual mouse food intake.

Procedure 5: Respiration

Equipment, software, and supplies

• Individual metabolic cages, with food provided ad libitum in a feeder placed at the bottom of the cage. The cage does not contain any bedding nor enrichment/toys (Columbus Instruments, Comprehensive Lab Animal Monitoring System (CLAMS), Columbus, OH, USA)

Steps

1. Test mice are placed in individual metabolic cages for a 48h time period; the first 24h are used for adaptation, and the second 24h period for data measurement.

Procedure 6: Oral glucose tolerance test (OGTT) and insulin test

Fasting (duration): Fasted overnight prior to test

Equipment, software, and supplies

• Glucometer

Steps

1. Mice are fasted overnight.
2. Mice are weighed and a small blood sample, taken from a tail tip incision, is used to measure the fasted glucose baseline (GTT time 0, 0 minutes). A 20% glucose oral gavage solution is given at 10 mL per kg of body weight, and tail-tip blood samples obtained at 15, 30, 45, 60, 90, 120, 150 and 180 minutes after gavage.
3. Blood samples are taken at 0, 15 and 30 minutes to measure insulin levels. The OGTT and insulin test are carried out at 17 weeks of age.

Procedure 7: Blood pressure

Equipment, software, and supplies

• Blood pressure analysis system, system II, tail-cuff system (Visitech Systems, BP-2000, Apex, NC, USA)

Steps

1. Non-invasive blood pressure is measured over a 4-day period, the first two days are considered as adaptation to the apparatus, and only measurements over the last two days are used for analysis. The measurements recorded such as systolic and diastolic blood pressures and heart rate are averaged across both days. Outlier readings are discarded but outlier mice are retained for further studies.
2. All measurements are recorded at 19 weeks of age.

Procedure 8: Cold response test

Environmental Conditions

Holding Area and/or Sample Collection Area

Description of Holding Enclosure: Pre-chilled standard housing cages in a room maintained at a temperature of 4°C, with only simple woodchip bedding, without supplement (e.g., tissue paper).

Max # Animals Per Enclosure: 1

Temperature (°C): 4

Steps

1. Mice are placed in pre-chilled cages (4°C) and body temperature (rectal) measured at 0, 1, 2, 3, 4, 5 and 6 hours. All mice are then returned to their normal housing cages. All measurements are carried out at 21 weeks of age.

Procedure 9: Basal activity

Equipment, software, and supplies

• Individual housing cages, woodchip bedding retained but tissue bedding removed as it interfered with laser detection
• Laser detection grids (TSE Systems, Bad Homburg, Germany)

Steps

1. Mice in individual housing cages are placed in laser detection grids, and movement detected as beam breaks over a 48h period, for horizontal (x-y, ambulation) and vertical (z, rearing) movement. The study starts at 10 am, with only the night cycle (7 pm to 7 am with 30 min of both dawn and dusk) is used for measurement collection. The study is carried out at 23 weeks of age.

Procedure 10: Treadmill

Equipment, software, and supplies

• Metabolic and Telemetric Modular Treadmill, treadmill tilt angle is set to 10 degrees for mice on chow diet (CD), and 0 degrees for mice on high-fat diet (HFD) (Columbus Instruments, Columbus, OH, USA)

Steps

1. Mice are introduced to the VO_2max treadmill, and allowed a 15 min habituation period, with only the last 2-min time period being used for analysis of basal activity. During this period of time the treadmill is left immobilized, and most mice spent this time exploring the device.
2. The treadmill is equipped with a grid that delivers mild electric shocks (0.1 mA to 0.3 mA) when mice stop running.
3. The treadmill is started at a speed of 4.8 m/min, the speed is then increased over a 60-second period, to 9 m/min for 4 minutes at that pace. The speed is then increased to 12 m/min over a 12-second time period for 4 min at that pace, followed by a speed increase to 15 m/min over 60 seconds for 4 min; the speed is then increased continuously by 0.015 m per second (or +0.0 m/min) thereafter until the end of the experiment at 63.5 min, 1354.5 m, or when the mouse is exhausted.
4. The endpoint of the experiment is determined when at least one of the three following conditions is achieved: 1) VO_{2 max} is reached (i.e. VO₂ levels fail to increase despite increasing running velocity or RER is above 1). 2) A mouse receives more than 5 electric shocks per minute in two consecutive minutes (it is considered that the animal has reached exhaustion). 3) A mouse completes the maximal running distance.
5. The distance, maximum VO_2max and maximum RER are measured. Maximum values are only recorded that are consistent across multiple measurements, not single-measurement spikes, which are disregarded.
6. Following the wheel activity exercise mice are exposed to a second treadmill test. Three mice completed the second test, the test was stopped for all other mice once they had reached exhaustion, such as falling off the treadmill and an inability to recover and continue running.
7. The test is carried out immediately after the 2-day basal activity assessment at 23 weeks of age.

Procedure 11: Activity wheel exercise

Equipment, software, and supplies

• Open-air cages, with access to either chow or high-fat diets according to test cohort
• Activity wheel (Bioseb, BIO-ACTIVW-M, Vitrolles, France)

Steps

1. Immediately after the treadmill test, test mice are placed in individual open-air cages with ad libitum access to activity running wheels, for a period of up to 10 days. The final 24h of wheel activity are recorded.
2. After the 10th day of wheel exercise, at about 25 weeks of age, test mice are exposed to a further treadmill test.
3. Following the second treadmill test mice are returned to their standard housing cages.

Procedure 12: Sacrifice and blood/tissue collection

Fasting (duration): Overnight fast prior to sacrifice

Equipment, software, and supplies

• Lithium-heparin (LiHep) coated tubes
• EDTA-coated tubes
• Dissection kit

Reagents and solutions

• Isoflurane anesthesia
• Phosphate buffered saline (PBS)

Steps

1. Mice are killed from 9-10:30am, at 29 weeks of age, with isoflurane anesthesia administration and by a complete blood draw (~1 mL) from the vena cava, followed by perfusion with PBS.
2. Half the collected blood is placed in lithium-heparin (LiHep)-coated tubes and other half in EDTA-coated tubes, both samples tubes shaken and stored on ice; the liver is then excised, the gallbladder removed, and the liver cut into small pieces and stored frozen in liquid nitrogen until required for analysis of metabolites.
3. The LiHep blood sample, taken for plasma analysis, is centrifuged at 4500 rpm for 10 min at 4°C, and stored in liquid nitrogen. Whole blood taken for cellular analysis is processed immediately after sacrifice, i.e. 1-2 hrs on ice.

Procedure 13: Organ weights

Equipment, software, and supplies

• Balance scale

Steps

1. Following sacrifice, the skeletal and soleus muscles, heart, kidney, liver, and the renal fat pad and subcutaneous adipose tissues are excised and weighed.