Cheverud1 project protocol

Diet-induced obesity and diabetes in 19 LGXSM recombinant inbred strains of mice   (2004)

Cheverud JM
With: Hrbek T, de Brito RA, Wang B, Pletscher LS, Ehrich TH, Kenney JP, Semenkovich CF

See also: Cheverud1 animal documentation


Cheverud1_Protocol

Project protocol — Contents
Workflow and sampling
Equipment
Reagents, supplies, and solutions
Procedures
Data
References



Workflow and sampling

Workflow

Age (wk)
Category
Procedure performed
Data collected
1-20
Growth curve Weekly bw measurements Weekly body weight (bw)
10
Blood chemistry Glucose assay, glucose tolerance Serum glucose
20
Blood chemistry Glucose assay, glucose tolerance Serum glucose
20-30
Body composition and organ metrics, blood chemistry
Necropsy and organ weights, glucose and lipid assays, hormone assay
Serum glucose, cholesterol, triglycerides, HDL, insulin, and leptin; heart, fat pad, spleen, liver, and kidney weights

Equipment

Top loading balance: for measurement of body weights

Blood glucose meter Glucometer Dex (Bayer)

Reagents, supplies, solutions

Composition of low- and high-fat diet (Harlan Teklad catalog #TD88137) and the low-fat diet (Research Diets catalog #D12284):

Components
Food type
(1 Kg) High fat diet (g)
(1 Kg) Low fat diet (g)
Casein Protein
195
197
Sugar Carbohydrate
341
307
Corn starch Carbohydrate
150
313
Cellulose Fiber
50
30
Corn oil Vegetable fat
-
58
Hydrogenated coconut oil Vegetable fat
-
7
Anhydrous milk fat Milk fat
210
-
Cholesterol
-
1.5
-
Energy from fat
-
42%
15%
Kilojoules per gram
-
18.95
16.99

Table 1. Composition of high- and low-fat diets as given in Cheverud et. al in Diabetes, volume 53, December 2004, page 3329.

10% glucose solution

HemoCue glucose system (HemoCue AB, Angelholm, Sweden)
HemoCue Beta-glucose analyzer (HemoCue) vs. Yellow Springs Instrument (YSI 2300 STAT; YSI) glucose oxidase analyzer

Sodium pentobarbital anesthetic solution

Procedures

I. Weekly body weight measurements

a. At 3-wk of age, while the mice are being weaned, the weanlings are randomly assigned to low- or high-fat diet.
b. While maintained in their respective dietary regime, the mice are also weighed weekly until 20-wk of age.

Parental strains: Large (LG/J) white mouse on the left and Small (SM/J) agouti mouse on the right (photo from The Cheverud Lab).

II. Blood glucose measurement and glucose tolerance test

a. At 10- and 20-wk of age, the mice are fasted for 4 hrs before blood is drawn to measure serum glucose.
b. After obtaining fasting glucose levels, the mice are then given intraperitoneal injection of 0.01 mL of 10% glucose solution per gram body weight.
c. Blood samples are taken at 15, 30, 60, and 120 min for glucose tolerance test.
d. A blood glucose meter is used to measure glucose levels in a drop of blood sample according manufacturer's instruction.

III. Necropsy, body composition, blood chemistry, and hormone measurements

a. Between 20-30-wk of age, the mice are again fasted for 4 hrs and prepared for necropsy.
b. For restraint, mice are given i.p. injection of sodium pentobarbital before obtaining blood via cardiac puncture, which is a terminal procedure.
c. Tail lengths are measured as an added indicator of growth (skeletal).
d. The thoracic and abdominal cavities are opened for the dissection and subsequent weighing of the heart, spleen, liver, and kidneys.
e. The different fat pads are dissected and weighed (see Figure)

Note: To facilitate the dissection of the gonadal fat pads, the entire uterus and ovaries or testes can be removed from their attachments to the pelvic floor. Likewise, the entire intestinal loop can also be removed from its attachment to the root of the mesentery, and then straightened and unwound for the dissection of the mesenteric fat pad. In contrast, both the inguinal and the perirenal fat pads may be dissected in situ.

f. Collected blood is centrifuged for the separation of plasma, and its storage- to be analyzed at a later date for blood lipids (free fatty acid, cholesterol, HDL, and triglycerides) and hormones (insulin and leptin).

Data collected by investigator

Weekly body weight (from 1-wk to 20-wk of age), 10 and 20-wk serum glucose plus glucose tolerance and glucose clearance (AUC), 20-30wk plasma lipid (cholesterol, HDL, triglycerides, and free fatty acid) levels, and hormone (insulin and leptin) levels, followed by necropsy body weight, organ (liver, spleen, kidney, heart) weights, fat pads (gonadal, inguinal, mesenteric, and perirenal) weights.

MPD calculated measurements: fat pad weight as % of body weight.

Definitions & formulas

AUC: Area under the curve (graph of blood glucose levels following i.p. injection of 10% glucose challenge, typically obtained at 15, 30, 60, and 120 min), which is indicative of a mouse's ability to clear glucose. Mice with low AUC reflect a greater efficiency in clearing glucose, as well as healthier insulin mobilization and sensitivity.

Glucose clearance: log10[baseline glucose (mg/dL) ÷ glucose (mg/dL) 2 hrs post-injection]

Total adiposity index = [total weight of isolated fat pads (g) ÷ necropsy body weight (g)]

Percent adiposity = [weight of isolated fat pad (g) ÷ necropsy body weight (g)] x 100

growth rate = log10[bw ÷ age] (g/wk)

pre-weaning = from birth to three weeks of age.

post-weaning = from 3 wks of age to 10 wks of age.

adult = 10 wks old and beyond.



References

    Cheverud JM, Pletscher LS, Vaughn TT, Marshall B. Differential response to dietary fat in large (LG/J) and small (SM/J) inbred mouse strains. Physiol Genomics. 1999 Jul 15;1(1):33-9. PubMed 11015559

    Cheverud JM, Vaughn TT, Pletscher LS, Peripato AC, Adams ES, Erikson CF, King-Ellison KJ. Genetic architecture of adiposity in the cross of LG/J and SM/J inbred mice. Mamm Genome. 2001 Jan;12(1):3-12. PubMed 11178736

    Ehrich TH, Kenney JP, Vaughn TT, Pletscher LS, Cheverud JM. Diet, obesity, and hyperglycemia in LG/J and SM/J mice. Obes Res. 2003 Nov;11(11):1400-10. PubMed 14627762

    Stylianou IM, Korstanje R, Li R, Sheehan S, Paigen B, Churchill GA. Quantitative trait locus analysis for obesity reveals multiple networks of interacting loci. Mamm Genome. 2006 Jan;17(1):22-36. Epub 2006 Jan 13. PubMed 16416088