Project protocol — Contents
Workflow and sampling
Equipment
Reagents, supplies, and solutions
Procedures
Data
References
Workflow
Mice acclimated to animal facility Unfasted mice tested for glucose glucose analyzer, reagent kits baseline glucose Mice fasted for 4-6 h, given 10% D-glucose i.p., and tail bled for glucose tolerance test (GTT); mice rested one week balance scale, needles and syringes, glucose analyzer, reagent kits GTT: blood glucose at 0, 30, 60, and 120 min; plasma insulin at 30 min only Mice fasted for 4-6 h, given regular human insulin i.p., and tail bled for insulin tolerance test (ITT); mice rested one week needles and syringes, glucose analyzer, reagent kits ITT: blood glucose at 0, 30, 60, and 120 min Mice weighed and anesthetized for densitometry Lunar densitometer, balance scale body weight and body composition Mice fasted for 4-6 h, weighed, sacrificed and blood collected from caudal vena cava for analysis balance scale, needles and syringes, dissecting kit, glucose analyzer, reagent kits body weight, glucose, total and free cholesterol, cholesterol ester, triglycerides, and BHB* Liver harvested, weighed, and stored at -80°C dissecting kit, balance scale, liquid nitrogen, freezer liver weight Liver processed for the extraction of hepatic lipids Extracted hepatic lipids analyzed for triglycerides reagent kits hepatic triglycerides *Ã-hydroxybutyric acid
⢠small animal densitometer (Lunar) for dual energy X-ray absorptiometry
⢠B-glucose analyzer (Hemocue)
⢠balance scale
â¢heating unit
⢠automatic timer
⢠small animal dissecting kit
⢠scintillation counterReagents, supplies, solutions Â
⢠needles and syringes
⢠liquid nitrogen
⢠cold phosphate buffered saline (PBS)
⢠chloroform
⢠petroleum ether
⢠potassium hydroxide (30% KOH solution)
⢠50% alcohol
⢠sodium dodecyl sulfate (SDS)
⢠copper tartrate
⢠regular human insulin (0.75 units/kg body weight, Eli Lilly and Co., Indianapolis, IN)
⢠commercial kits used to measure plasma concentrations of glucose (Sigma), free fatty acids (FFAs), cholesterol, triglycerides (Wako Chemicals USA, Inc.), and plasma insulin (Linco Inc)I. Glucose Tolerance Test (GTT)/Insulin Tolerance Test (ITT)
1. Baseline blood glucose is measured in unfasted mice using commercial kits (Sigma) according manufacturers instructions.
2. Mice fasted for 5 h are given an intraperitoneal injection of 10% D-glucose (0.75 g/kg body wt) for glucose tolerance test (GTT).
3. Blood (10 µL) is drawn from the tail vein at 0, 30, 60, and 120 min and assayed for glucose using a glucose meter (Hemocue, Mission Viejo, CA). 30 min after glucose administration, blood sample for plasma insulin is obtained, and approximately of blood is drawn from the tail vein at and assayed for blood glucose.
4. An extra sample of blood is taken at 30 min for an insulin test, analyzed by radioimmunoassay (Linco Inc) according manufacturers instructions.
6. Mice are allowed 1 wk to recover before insulin tolerance test (ITT) is conducted.
7. Mice fasted for 5 h are given an intraperitoneal injection of regular human insulin (0.75 units/kg body weight) for insulin tolerance test.
8. Blood (10 µL) is drawn from the tail vein at 0, 30, 60, and 120 min and assayed for plasma glucose as above.
9. Mice are allowed 1 wk to recover before body composition measurement is conducted.II. Measurement of percent body fat
1. Mice are fasted for 4-6 h prior to weighing.
2. Mice are anesthetized based on body weight.
3. Fully anesthetized mice are assessed for body fat (%) via dual energy X-ray absorptiometry or a small animal densitometer (DXA Lunar®).III. Measurement of plasma parameters
1. While mice are still under deep anesthesia, the visceral abdomen is exposed, and the caudal vena cava undermined for subsequent blood collections.
2. In the presence of an anti-coagulant, as much blood is drawn from the caudal vena cava, with eventual exanguination.
3. Blood samples are centrifuged and processed for plasma and subsequent blood chemistry assays.
4. The liver is harvested and saved (see below).
5. Plasma free fatty acids (FFA) are analyzed by an enzymatic method (Wako), while blood glucose is determined using a B-glucose analyzer (Hemocue, Ängelholm, Sweden).
6. Plasma beta-hydroxybutyrate (BHB) concentration is analyzed at the Core Laboratory of the General Clinical Research Center at Washington University.
7. Plasma triglycerides, total cholesterol, and free cholesterol are determined using commercial kits according manufacturers instructions.IV. Measurement of liver parameters
1. After livers are excised, washed in cold PBS, weighed, and cut into pieces, they are then frozen in liquid nitrogen, and stored at -80°C until further analysis.
2. Slices (~250 mg) of fully thawed liver are digested in a potassium hydroxide solution (30%) at 95°C for 30 min.
3. Digested liver samples are then saponified in 30% KOH:50% alcohol at 95°C for 3 h.
4. Nonsaponifiable lipids are initially extracted with petroleum ether.
5. The remaining sample solution containing the saponified lipids is then acidified with sulfuric acid, and the fatty acids are finally extracted with petroleum ether.
6. Extracted hepatic lipids are assayed for triglycerides (TG) by an enzymatic method (Wako), and are expressed as milligrams of lipid per gram of protein.
7. Total hepatocyte protein content is measured using a modification of the Lowry method of protein determination with the addition of sodium dodecyl sulfate (SDS) in the alkali reagent and an increase in the amount of copper tartrate reagent (see Markwell et al., reference below).Data collected by investigator
- Unfasted baseline blood glucose
- Blood glucose (fasted 5 h) levels: glucose tolerance test (GTT 0, 30, 60, and 120 min post-glucose injection)
- Plasma insulin levels (GTT 30 min post-glucose injection)
- Blood glucose (fasted 5 h) levels: insulin tolerance test (ITT, 0, 30, 60, and 120 min)
- Body composition including percent body fat and body weight.
- Blood lipids including total cholesterol, free cholesterol, cholesterol esters, free fatty acids, plasma phospholipids, and triglycerides
- Blood β-hydroxybutyrate (BHB) keytone body
- Liver weight and hepatic triglycerides levels
Definitions and calculations
The area under the glucose and insulin curves (AUC) during the GTT and ITT are calculated using the formula:
AUC = 0.25 (fasting value) + 0.5 (0.5 h value) + 0.75 (1 h value) + 0.5 (2 h value)
References
Carr TP, Andresen CJ, Rudel LL. Enzymatic determination of triglyceride, free cholesterol, and total cholesterol in tissue lipid extracts. Clin Biochem. 1993 Feb;26(1):39-42.
PubMed 8448837 Lin X, Schonfeld G, Yue P, Chen Z. Hepatic fatty acid synthesis is suppressed in mice with fatty livers due to targeted apolipoprotein B38.9 mutation. Arterioscler Thromb Vasc Biol. 2002 Mar 1;22(3):476-82.
PubMed 11884293 Markwell MA, Haas SM, Bieber LL, Tolbert NE. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206-10.
PubMed 98070