7. Regularly monitor body weight and food intake (twice per
week). Food intake per animal can be estimated on a per-cage
basis, from the actual amount of food consumed by the animals, its caloric equivalence, and the number of animals
per cage.
8. It is advisable to perform the following analyses and tests in live
animals, as in [24]:
l
Body composition using suitable magnetic resonance equipment (e.g., EchoMRI-700TM analyzer).
l
Energy expenditure and respiratory exchange ratio (RER)
through indirect calorimetry (e.g., using PhenoMaster Indirect Calorimetry System) and spontaneous locomotor activity through a motion sensor coupled to the indirect
calorimetry equipment.
l
A glucose tolerance test and the estimation of the surrogate
insulin resistance indexes. For this, intraperitoneally administer a glucose load (2 g per kg of body weight in 100 μL of
sterile saline) to mice after a 6-h fast (from 6 am to 12 pm).
Collect blood samples (one drop) from the tail before the
glucose load (time 0) and at 30, 60, 120, and 180 min
thereafter and measure the levels of glucose with a glucometer. In addition, at time 0 collect a blood sample (maximum 200 μL) to measure the levels of insulin and free fatty
acids (by specific ELISA and enzymatic assays, respectively)
for the estimation of the surrogate insulin resistance
indexes.
l
The homeostatic model assessment for insulin resistance
(HOMA-IR) score is calculated from fasting insulin and
glucose concentrations as follow: HOMA-IR ¼ insulin
(in μU/L) Â glucose (in mmol/L)/22.5.
l
The revised quantitative insulin sensitivity check index
(R-QUICKI) is calculated as follow: R-QUICKI ¼ 1/[log
glucose (in mg/dL) + log insulin (in μU/mL) + log NEFA
(in mEq/L)].
9. On day 70 (week 10 of treatment), proceed to the euthanization and tissue collection, within the first 3 h of the light cycle
(distribute the animals over several days).
10. Anesthetize the animal with isoflurane [25]. For this, place the
animal in the anesthetic induction chamber, and set the vaporizer at 4–5% isoflurane. When the animal loses consciousness,
remove it from the chamber and place a mask on it, with the
vaporizer at 1–2% isoflurane and oxygen at 100%
(maintenance).
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Joan Ribot et al.
week). Food intake per animal can be estimated on a per-cage
basis, from the actual amount of food consumed by the animals, its caloric equivalence, and the number of animals
per cage.
8. It is advisable to perform the following analyses and tests in live
animals, as in [24]:
l
Body composition using suitable magnetic resonance equipment (e.g., EchoMRI-700TM analyzer).
l
Energy expenditure and respiratory exchange ratio (RER)
through indirect calorimetry (e.g., using PhenoMaster Indirect Calorimetry System) and spontaneous locomotor activity through a motion sensor coupled to the indirect
calorimetry equipment.
l
A glucose tolerance test and the estimation of the surrogate
insulin resistance indexes. For this, intraperitoneally administer a glucose load (2 g per kg of body weight in 100 μL of
sterile saline) to mice after a 6-h fast (from 6 am to 12 pm).
Collect blood samples (one drop) from the tail before the
glucose load (time 0) and at 30, 60, 120, and 180 min
thereafter and measure the levels of glucose with a glucometer. In addition, at time 0 collect a blood sample (maximum 200 μL) to measure the levels of insulin and free fatty
acids (by specific ELISA and enzymatic assays, respectively)
for the estimation of the surrogate insulin resistance
indexes.
l
The homeostatic model assessment for insulin resistance
(HOMA-IR) score is calculated from fasting insulin and
glucose concentrations as follow: HOMA-IR ¼ insulin
(in μU/L) Â glucose (in mmol/L)/22.5.
l
The revised quantitative insulin sensitivity check index
(R-QUICKI) is calculated as follow: R-QUICKI ¼ 1/[log
glucose (in mg/dL) + log insulin (in μU/mL) + log NEFA
(in mEq/L)].
9. On day 70 (week 10 of treatment), proceed to the euthanization and tissue collection, within the first 3 h of the light cycle
(distribute the animals over several days).
10. Anesthetize the animal with isoflurane [25]. For this, place the
animal in the anesthetic induction chamber, and set the vaporizer at 4–5% isoflurane. When the animal loses consciousness,
remove it from the chamber and place a mask on it, with the
vaporizer at 1–2% isoflurane and oxygen at 100%
(maintenance).
408
Joan Ribot et al.