Manipulation of Mouse Metabolism.

In a typical experiment, animals of different genotypes or in different experimental conditions are challenged with one or more experimental diets, and their biological responses under these diets are compared to those of animals on a control chow. While it is not possible to outline a single protocol to cover all such experiments, key parameters to consider include using appropriate numbers of age- and gender-matched mice (see Critical Parameters for more details) and maintaining the diets for sufficient times to induce the desired responses. Typical times required for the induction of various pathologies are indicated in Table 29B.5.1.

Most of the diets described in Table 29B.5.1 are generally formulated by commercial animal food suppliers. Alternatively, experimenters with specific requirements may want to have custom diets formulated either by commercial food suppliers that offer this service or by academic facilities that specialize in animal nutrition. In any case, it should be kept in mind that most of these diets are only partially defined diets, and variations in the composition of certain constituents can modify the biological response in vivo. For example, a high-fat diet is most commonly made from animal fat using lard, but the nature of lard triglycerides can vary according to the specific origin and can thereby affect the biological outcome.

BASIC PROTOCOL 2

ADMINISTRATION OF A TEST COMPOUND THROUGH THE DIET

Incorporating a compound in the diet to test for its physiological actions is an easy and efficient way to achieve reproducible daily exposure with minimal stress to the animals. The type of diet in which the compound is mixed should be chosen, on the one hand, according the appropriate diet for the purpose of the study and, on the other hand, according to the chemical properties and bioavailability of the compound itself. Indeed, the polarity of the compound can influence its bioavailability. For example, in the case of a lipophilic molecule, the fat content of the diet can modify its intestinal absorption by increasing its incorporation into lipid micelles. In addition, technical considerations relating to the amount of compound to incorporate, its polarity, and the type of diet chosen should be evaluated in order to achieve a homogeneous incorporation, which will ensure constant and homogeneous dosing of the animals (see Critical Parameters and Fig. 29B.5.3).

Materials

Appropriate mouse strain (e.g., C57BL/6 or see Commentary)

Mouse diet (standard or high-fat diet) in powdered form

Test compound

Vehicle to dissolve compound (if required)

Group housing cages for mice

Scale with 0.1 g accuracy (to weigh food and mice)

Container to accommodate mice during weighing

Scale with 0.1 mg accuracy (to weigh compound)

Mortar and pestle for grinding the compound (if required)

Large container for mixing diet and test compound or automated mixing machine

Commercial pelleting machine (optional)

Desiccator (optional)

Determine daily food intake

The accuracy of the doses administered through food relies on the correct measurement of food intake (FI), which directly controls the amount of compound ingested. Therefore, FI should be evaluated, always housing the same number of animals per group.

1. At a given hour of the day, change mice to clean cages and weigh the amount of food given.

2. Two to seven days later, at the same hour of the day, weigh the food remaining, including any chunks of pellets in the cage.

3. Calculate FI in g/mouse/day according to the formula:

Equation 29B.5.1

where w₀ is the weight (g) of the amount of food given;

w_nd is weight (g) of the food remaining in the cage at the end of the feeding period;

n_d is the number of days over which FI is calculated;

n_m is the number of mice per cage.

4. Repeat the measurement every two days during the first week of treatment to verify that the compound does not cause food aversion, and on a weekly basis thereafter.

Calculate test compound dose

A test compound is generally administered at a constant dose normalized to body weight (mg compound/kg body weight). Since accomplishing this for each individual animal would require individual housing and preparation of a diet with a concentration of compound adapted to each mouse, a widely accepted simpler approach consists of using the average body weight (BW) and food intake (FI) as an estimate for each animal. Alternatively, in the experimental case where both the diet and the treatment are expected to minimally affect body weight, the compound can be administered at a constant concentration in the diet. The concentration should be calculated the same as for a constant dose, but only once, using the BW and FI calculated before the beginning of the treatment or from estimates of these parameters derived from pilot studies performed in the exact same experimental setting.

5a. To calculate test compound as a constant dose in diet (mg/kg body weight): Adjust the quantity of compound to incorporate into the diet (QCD) given on a weekly basis by calculating for week n the QCD_n in mg compound/g diet according to the following equation:

Equation 29B.5.2

where LE is the desired level of exposure (mg/kg BW/day);

BW_n−1 is the average body weight (kg) from week the previous week (n-1);

FI_n−1 is the food intake (g/mouse/day) from week the previous week (n-1).

The incorporation of the compound into the diet requires weekly adaptation to adjust for variations in FI and BW. Thus, these parameters should be recorded on at least a weekly basis, at a controlled time of day, as described in steps 1 to 4.

5b. To calculate test compound as a constant concentration (mg/kg diet): Calculate the concentration of test compound to be incorporated into the diet, using Equation 29B.5.2 and the BW and FI calculated before the beginning of the treatment or from estimates of these parameters derived from pilot studies performed in the exact same experimental setting.

After validating the stability (in the diet) of the compound to be incorporated, this alternative has the advantage of allowing the preparation of a large stock of pellets for long treatment periods, and therefore it has the potential to outsource the pelleting process.

Prepare pellets containing test compound

6a. To incorporate a compound into a high-fat diet: Mix the compound directly with the high-fat diet powder, using either manual mixing in a large container or an automated mixing machine.

The high lipid content of high-fat diet enables direct compaction.

Incorporating low amounts (<100 mg/kg) of compound will require using an appropriate vehicle to ensure homogeneous incorporation (see Critical Parameters and Fig. 29B.5.3 for details).

6b. To incorporate a compound into a standard diet: Incorporate 600 ml/kg autoclaved distilled water in standard chow diet powder. Mix in the powdered compound, using either manual mixing in a large container or an automated mixing machine.

Adding the water enables compaction.

Incorporating low amounts (<100 mg/kg) of compound will require using an appropriate vehicle to ensure homogeneous incorporation (see Critical Parameters and Fig. 29B.5.3 for details).

7. Form pellets manually (by tightly packing the mixture) or using a commercial pelleting machine (recommended).

Alternately, animals can be fed the powdered diet (see step 9 annotation).

8. Store high-fat diet pellets up to 2 months at 4°C (or frozen according to the manufacturer’s instruction) until use. Air-dry chow diet pellets in a desiccator or in a ventilated hood for at least 48 hr and store up to 6 months at room temperature or according to the stability of the compound (see Critical Parameters).

Administer test compound in diet

9. Give a large excess of pellets (at least 20% more than the amount required according to FI on week n-1) in a clean cage, at a weekly frequency (minimum).

Although animals can be fed the powdered diet from step 1, it is essential that specialized equipment be used, as mice will spill large quantities of food and rapidly contaminate open troughs. For example, special troughs with grids have been designed to limit these problems, and one is illustrated in Figure 29B.5.1. To avoid these problems entirely, it is best to restrict this feeding of powdered diet to specialized applications where powdered diet is absolutely required (e.g., monitoring food consumption in metabolic cages; see Basic Protocol 4 of UNIT 29B.1).

Figure 29B.5.1 Example of a food trough for powdered diet. Powdered diet is placed in the barrel-shaped container and the metal grid (which limits food spoiling and contamination) is positioned on top of the diet and secured with screws and a grid holder. Mice access food via the small holes of the grid.

BASIC PROTOCOL 3

CALORIC RESTRICTION

Caloric restriction (CR) is the main nongenetic manipulation known to extend lifespan in a wide variety of animal models. Although various CR strategies have been experimentally tested, with different levels of feeding restriction, CR is typically defined as a 33% to 50% decrease in total caloric intake. In order to maintain a balanced nutritional supply, diet composition is unchanged to ensure that all sources of nutrients, vitamins, and minerals are provided, and only the quantity of food the animals can access daily is held at levels lower than ad libitum.

The altered metabolic rate induced by this dietary manipulation has been demonstrated to produce a metabolic profile desirable for treating diseases of aging in both animal models and humans (e.g., metabolic disorders or neurodegeneration; Koubova and Guarente, 2003; Masoro, 2005). CR has also been shown to reduce the incidence and progression of spontaneous and induced tumors (Weindruch and Walford, 1988). It is thus not surprising that caloric-restricted mice are a model used extensively to understand the fundamental mechanisms underlying these prevalent human pathologies, and that developing pharmacological caloric restriction mimetics is a current research strategy for antineurodegenerative and anticancer interventions (Roth et al., 2005; Elliott and Jirousek, 2008).

An easily applicable method for restricting caloric intake is to feed the CR group with the same diet as a control group fed ad libitum, but to fast them for 24 hr on alternate days. This method leads to a 30% to 40% decrease of the global caloric intake (Weindruch et al., 1986). However, it generates cycles of lipolysis and fat storage that are not representative of a constant reduction in caloric intake.

The most reliable method for restricting caloric intake consists of pair-feeding CR mice at 50% to 67% of a control group fed ad libitum by restraining their daily food intake. A caloric restriction of 40% is recommended (i.e., pair-feeding at 60% of the ad libitum control). Ideally, mice should be housed in individual cages. If this is not possible, they should at least be housed at the same number of animals per cage. The quantity of food to be administered to the CR group should be evaluated daily as described in the steps below.