5. Liquid nitrogen.
6. 4% paraformaldehyde in 0.1 M phosphate buffer a pH 7.4, to
fix fresh tissue biopsies.
3 Methods
3.1 Model to Assess
the Capacity
of Carotenoids
to Counteract
the Development
of Metabolic
Alterations
and Obesity Induced
by an Obesogenic Diet
Here we propose a protocol in mice to investigate the potential of a
given carotenoid, carotenoid mixture, or carotenoid-rich extract to
counteract the development of obesity and prevent the metabolic
alterations caused by feeding mice a moderate high-fat diet (45% of
caloric content) (see Note 1) for 10 weeks (see Note 2). If knockout
mouse models deficient for carotenoid dioxygenases (see Note 3)
are used, this may allow dissecting the effects of parent carotenoids
apart from the effects of derived apocarotenoid products. For
instance, as seen in [12], if an antiobesogenic effect of the tested
carotenoid is observed in wild-type control mice but is lacking in
the matched knockout, an involvement of carotenoid-dioxygenase
products could be suspected instead of the parent carotenoid.
1. Perform the experiments with 4–6-week-old (see Note 2)
C57BL/6J (see Note 4) male (see Note 5) mice.
2. Animals must be kept confined and handled according to existing ethical guidelines for the use and care of laboratory animals
(see Note 6).
3. Divide mice by weight-matching into four experimental groups
and house 3–4 mice per cage in a controlled temperature and
humidity room with 12-hour light–dark cycle and free access to
food and water (10 animals/group suggested, or 3–4 cages/
group). Feed all mice with a defined low-fat low-sucrose (LFD)
diet with 10% energy as fat and 7% energy as sucrose.
4. After 1 week (day 0 of the experiment), switch three experimental groups to a defined high-fat medium-sucrose (HFD)
diet with 45% energy as fat and 17% energy as sucrose and
continue feeding the remaining experimental group with the
LFD diet (see Notes 7 and 8).
5. At day 0, start the treatment. Give the mice of the LFD-fed
group and of one HFD-fed group the vehicle (olive oil); these
will serve as, respectively, LFD-fed and HFD-fed reference
groups. Treat the other two HFD-fed groups with an emulsion
of the selected pure carotenoid, mix or extract (see Notes 9 and
10) at two different doses (see Note 11). Conduct the treatments daily by oral administration, with the aid of a pipette
(20 μL), more or less at the same time during the day. Prepare
the carotenoid emulsions every day to prevent degradation.
6. The duration of the nutritional and treatment challenge is
suggested to be 10 weeks (see Note 2).
Mouse Models to Study Antiobesogenic Effects of Carotenoids
407
6. 4% paraformaldehyde in 0.1 M phosphate buffer a pH 7.4, to
fix fresh tissue biopsies.
3 Methods
3.1 Model to Assess
the Capacity
of Carotenoids
to Counteract
the Development
of Metabolic
Alterations
and Obesity Induced
by an Obesogenic Diet
Here we propose a protocol in mice to investigate the potential of a
given carotenoid, carotenoid mixture, or carotenoid-rich extract to
counteract the development of obesity and prevent the metabolic
alterations caused by feeding mice a moderate high-fat diet (45% of
caloric content) (see Note 1) for 10 weeks (see Note 2). If knockout
mouse models deficient for carotenoid dioxygenases (see Note 3)
are used, this may allow dissecting the effects of parent carotenoids
apart from the effects of derived apocarotenoid products. For
instance, as seen in [12], if an antiobesogenic effect of the tested
carotenoid is observed in wild-type control mice but is lacking in
the matched knockout, an involvement of carotenoid-dioxygenase
products could be suspected instead of the parent carotenoid.
1. Perform the experiments with 4–6-week-old (see Note 2)
C57BL/6J (see Note 4) male (see Note 5) mice.
2. Animals must be kept confined and handled according to existing ethical guidelines for the use and care of laboratory animals
(see Note 6).
3. Divide mice by weight-matching into four experimental groups
and house 3–4 mice per cage in a controlled temperature and
humidity room with 12-hour light–dark cycle and free access to
food and water (10 animals/group suggested, or 3–4 cages/
group). Feed all mice with a defined low-fat low-sucrose (LFD)
diet with 10% energy as fat and 7% energy as sucrose.
4. After 1 week (day 0 of the experiment), switch three experimental groups to a defined high-fat medium-sucrose (HFD)
diet with 45% energy as fat and 17% energy as sucrose and
continue feeding the remaining experimental group with the
LFD diet (see Notes 7 and 8).
5. At day 0, start the treatment. Give the mice of the LFD-fed
group and of one HFD-fed group the vehicle (olive oil); these
will serve as, respectively, LFD-fed and HFD-fed reference
groups. Treat the other two HFD-fed groups with an emulsion
of the selected pure carotenoid, mix or extract (see Notes 9 and
10) at two different doses (see Note 11). Conduct the treatments daily by oral administration, with the aid of a pipette
(20 μL), more or less at the same time during the day. Prepare
the carotenoid emulsions every day to prevent degradation.
6. The duration of the nutritional and treatment challenge is
suggested to be 10 weeks (see Note 2).
Mouse Models to Study Antiobesogenic Effects of Carotenoids
407