Procedures

Procedure 1: Diet administration

Reagents and solutions

• Standard chow diet (Purina LabDiet, 5010, St. Louis, MO, USA)
• High-fat High-sucrose (HFHS) diet (Research Diets, D12331, New Brunswick, NJ, USA)

Steps

1. All mice are fed the chow diet until 5 weeks of age.
2. At 5 weeks, randomly selected males of each of the two strains are fed the HFHS diet until the end of the study, and the rest are continued to be fed the chow diet.

Procedure 2: Body weight and size determination and anesthetization

Fasting (duration): Overnight fast

Equipment, software, and supplies

• Balance scale
• Measuring ruler

Reagents and solutions

• Avertin, 0.8 mg/g of 2,2,2,-tribromoethanol in tertiary-amyl alcohol (Fisher Scientific, Pittsburgh, PA, USA)

Steps

1. Mice at 21 weeks of age are moved to clean cages, fasted overnight, weighed, and then anesthetized via intraperitoneal injection with Avertin. Nose to anus length is measured to calculate BMI.

Procedure 3: Blood glucose

Equipment, software, and supplies

• Glucometer (Lifescan Inc., One Touch Ultra, Milipitas, CA, USA)

Steps

1. Blood glucose is measured from the tail vein using a glucometer immediately after mice are anesthetized.

Procedure 4: Liver extraction and weight

Equipment, software, and supplies

• Balance scale

Reagents and solutions

• Liquid nitrogen

Steps

1. After euthanization, the liver is extracted and weighed. Liver samples are kept frozen in liquid nitrogen until required for analysis.

Procedure 5: Plasma preparation

Equipment, software, and supplies

• Plasma separation tube with gel barrier (Statspin, Westwood, MA, USA)
• Heparinized micro-hematocrit capillary tube (Fischer Scientific, Pittsburgh, PA, USA)

Steps

1. Blood from the orbital sinus is drawn into a separation tube with gel barrier using a heparinized micro-hematocrit capillary tube.
2. Plasma is isolated and transferred to a new tube, immediately frozen on dry ice and stored at -80 °C until required for analysis.

Procedure 6: Plasma cholesterol, triglycerides and insulin measurement and HOMA-IR

Definitions & Abbreviations: HOMA-IR # Homeostasis Model Assessment of Insulin Resistance

Equipment, software, and supplies

• ELISA (Mercodia, Winston-Salem, NC, USA)

Reagents and solutions

• Colorimetric reagents and standards (Pointe Scientific, Canton, MI, USA)

Steps

1. Plasma total cholesterol and triglycerides are measured using colorimetric reagents and standards as stated in the manufacturer's instructions.
2. Plasma insulin is measured with a mouse ultrasensitive insulin ELISA kit following manufacturer's instructions.
3. HOMA-IR is calculated as described previously by Wallace et al. (2004).

Additional ARRIVE Guidelines

Formulas & Derivations: HOMA-IR = [fasting insulin (mU/L) x fasting blood glucose (mmol/L)]/22.5

Procedure 7: Liver triglycerides

Equipment, software, and supplies

• Colorimeter

Reagents and solutions

• 3M KOH/65% ethanol
• GPO-Trinder reagent set (Pointe Scientific, Canton, MI, USA)

Steps

1. Liver samples are taken and approximately 100-200 mg of tissue are saponified in an equal volume (by weight) of KOH/ethanol solution, in order to convert triglycerides to glycerol and fatty acids.
2. Glycerol is measured colorimetrically with a GPO-Trinder reagent set, in order to quantify the triglyceride content.
3. Total liver triglycerides are estimated by multiplying the liver triglyceride content by total liver weight.