Nadeau2 project protocol

Diet effects on body weight and metabolic traits in the C57BL/6J-Chr#A/J/NaJ mouse chromosome substitution panel (males)   (2015)

Riordan JD, Croniger CM, Sinasac DS, Nadeau JH

Project protocol - Contents

Workflow and sampling

Data collected
At 5 wks of age, mice are weighed; randomly selected mice are switched to high-fat high-sucrose diet (HFHS) (controls remain on standard chow) for a duration of 16 wks Scales
Body weight
At 21 wks of age, mice are moved to a clean cage and fasted overnight (16h) -
3 Mice are weighed and anesthetized Scales Body weight
4 Body length measured Ruler Body length, body mass index (BMI)
5 Blood glucose measured Glucometer Blood glucose
6 Plasma isolated Heparinized micro-hematocrit capillary tubes -
7 Lipids, insulin levels measured - Plasma cholesterol and triglycerides; plasma insulin; HOMA-IR
8 Mice euthanized and liver harvested and prepared for analysis Scales Liver weight, liver triglycerides

Equipment and supplies

  • Scales
  • One Touch Ultra glucometer (Lifescan, Inc., Milipitas CA)
  • Plasma separation tubes with gel barrier (Statspin, Westwood MA)
  • Heparinized micro-hematocrit capillary tubes (Fisher Scientific, Pittsburgh PA)
  • ELISA (Mercodia, Winston-Salem NC)

Reagents and solutions

  • Standard chow (Purina 5010, Purina LabDiet, St. Louis MO)
  • High-fat high-sucrose diet (Research Diets D12331, New Brunswick NJ)
  • Avertin (2,2,2,-tribromoethanol in tertiary-amyl alcohol) Fisher Scientific, Pittsburgh PA
  • Colorimetric reagents and standards (Pointe Scientific, Canton MI)
  • GPO-Trinder reagent set (Pointe Scientific, Canton MI)
  • 3M KOH/65% ethanol
  • Dry ice
  • Liquid nitrogen

Procedure: Body size and anesthetization

    1. Mice (fasted overnight 16h) are weighed and anesthetized via intraperitoneal injection (i.p.) with 0.8 mg/g Avertin.
    2. Nose-to-anus length is measured and used for body mass index (BMI) calculation.

Procedure: Blood glucose

    1. Blood glucose is measured from the tail vein using a One Touch glucometer.

Procedure: Plasma preparation

    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 and immediately frozen on dry ice and stored at -80°C until analysis.

Procedure: Plasma lipids and insulin

    1. Plasma total cholesterol and triglycerides are measured with colorimetric reagents and standards according to manufacturer's directions.
    2. Plasma insulin is measured with a mouse ultrasensitive insulin ELISA according to manufacturer's directions.
    3. Homeostasis model assessment insulin resistance (HOMA-IR) is calculated as follows: HOMA-IR = [fasting insulin x fasting glucose]/22.5.

Procedure: Liver weight and triglycerides

    1. Livers are weighed and frozen in liquid nitrogen.
    2. Tissue (100-200 mg) is saponified in an equal volume by weight of 3M KOH/65% ethanol to convert triglycerides to glycerol and fatty acids.
    3. Glycerol is measured colorimetrically using a GPO-Trinder reagent set to quantify the triglyceride content.
    4. Total triglycerides are estimated by multiplying the liver triglyceride content by total liver weight.

Data collected by investigator

  • body weight
  • body length
  • BMI
  • blood glucose
  • plasma total cholesterol
  • plasma triglycerides
  • plasma insulin
  • liver weight
  • liver triglycerides
  • total liver triglycerides