Procedures

General Information for All Procedures: Fifteen cohorts of nine inbred mouse strains are fed either a CD or HFD diet in total, they are separated randomly into groups of five mice of each sex, for each diet. Strains are entered into the phenotyping program randomly, and are entered in a staggered entry, typically by 1 to 2 weeks.

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 of fat, 20% kCal of protein, and 20% kCal of carbohydrates (Harlan, 06414, Indianapolis, IN, USA)

Steps

1. All mice are fed a chow diet until 8 weeks of age.
2. At 8 weeks of age, half the mice are 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 of test mice are recorded weekly from 8 to 21 weeks of age.
2. In addition to weekly recordings, body weights are measured before each phenotypic experiment at 16 and 19 weeks of age, as well as at sacrifice (21 weeks).

Procedure 3: Body composition and weight by Echo-MRI

Definitions & Abbreviations: MRI # Magnetic Resonance Imaging

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 of mice are assessed by Echo-MRI at 14 weeks of age.
2. In addition to body weight recordings as recorded by balance scale (see above body weight procedure), body weight was also measured by Echo-MRI at 14 weeks of age.

Procedure 4: Respiration and food intake

Equipment, software, and supplies

• Comprehensive Laboratory Animal Monitoring System (Oxymax/CLAMS) (Columbus Instruments, Columbus, OH, USA)

Steps

1. Mice at 14 weeks of age 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 acquisition. This includes measurements such as the volume of oxygen inhaled, the volume of carbon dioxide exhaled and the derived parameters of these values, the respiratory exchange ratio (RER).
2. The daily food intake is also measured over the 48h period. The test is carried out at 14 weeks of age.

Procedure 5: Oral glucose tolerance test (OGTT) and insulin test

Fasting (duration): Fasted overnight prior to test

Equipment, software, and supplies

• Glucometer

Steps

1. Following an overnight fast, 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; following which tail-tip blood samples are obtained at 15, 30, 45, 60, 90, 120, 150 and 180 minutes after gavage.
2. Blood samples are taken at 0, 15 and 30 minutes to measure insulin levels. The OGTT and insulin test are carried out at 16 weeks of age.

Procedure 6: Cold response test

Equipment, software, and supplies

• Rectal thermometer

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.

Max # Animals Per Enclosure: 1

Temperature (°C): 4

Steps

1. Mice are placed in pre-chilled cages and basal 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 18 weeks of age.

Procedure 7: Basal activity test

Equipment, software, and supplies

• Individual housing cages, woodchip bedding retained and food and water provided as standard
• 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 separate measurements for the night cycle (defined as 7 pm to 7 am, with 30 min of both dawn and dusk), as well as for the day cycle (defined as 7 am to 7 pm), being acquired respectively for both horizontal and vertical movements of test mice.

Procedure 8: Treadmill (VO_2max) test

Equipment, software, and supplies

• Metabolic and Telemetric Modular treadmill, treadmill tilt angle is set to 10° for mice on chow diet (CD), and 0° 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 pre-treadmill activity. During this period of time the treadmill is left immobilized, and most mice spent this time exploring the device.
2. The treadmill is started at a speed of 4.8 m/min, the speed is then increased over a 60 s period, to 9 m/min for 4 minutes at that pace. The speed is then increased to 12 m/min over a 12 s time period for 4 min at that pace, followed by a speed increase to 15 m/min over 60 s for 4 min; the speed is then increased continuously by 0.015 m per second² (or +0.9 m/min²) thereafter until the end of the experiment at 63.5 min, 1354.5 m, or when the mouse is exhausted.
3. The endpoint of the experiment is determined when either the mice are exhausted or when physical constraints are found to be too high. The distance, maximum VO₂ and maximum RER are recorded. Only those maximum values are considered that are consistent across multiple measurements, and not single-measurement spikes which are discarded for the purposes of this test. Mice are tested at 19 weeks of age.

Procedure 9: Sacrifice, blood/tissue collection and lipid analysis

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 sacrificed from 8:30-10:30am, at 21 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 sample tubes are 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 10: Organ weights and size

Equipment, software, and supplies

• Balance scale
• Ruler

Steps

1. Following sacrifice, the gastrocnemius muscle, heart, kidney, liver, spleen, cecum and the renal fat pad and subcutaneous adipose tissues are excised and weighed. In addition, the entire intestine is excised, and then laid along a straight line on a flat surface and the length measured.