Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Body size and anesthetization
• Procedure: Blood glucose
• Procedure: Plasma preparation
• Procedure: Plasma lipids and insulin
• Procedure: Liver weight and triglycerides
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	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
2	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
• HOMA-IR
• liver weight
• liver triglycerides
• total liver triglycerides