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6. Procedures: (Relate directly to the Specific Aims cited in 5).
1 .
Animals
Sprague-Dawley rats and C57BL/6J mice will be housed in
a controlled environment (24.C, 12 hours of light/24 hours).
A synthetic diet rich in glucose will be given as a single
daily meal from 0900 to 1200. Water will be available ad
libitum. The animals will be allowed to adjust to the
feeding schedule form at least 2 weeks before being used
for experiments.
Experiments with animals will conform with the
"Guiding Principles in the Care and Use of Animals!
approved by the Council of the American Physiological
Society and with federal laws and regulations.
2 .
Acute and chronic treatments of animals with hydroxy-
citrate (HC)
Acute administration of HC (0.5 mmole/kg) will be
performed by intraperitoneal or intravenous injection of a
stock solution of sodium salt adjusted to PH = 7.40.
Chronic treatment will be performed by intraperitoneal
injection one half hour before the daily meal or by mixing
HC with the diet mixture (50 mmoles/kg diet).
3 .
Effect of hydroxycitrate on brain acetylcholine
synthesis
Acetylcholine synthesis. will be assayed in brains of
rats and mice after chronic and acute treatment with HC.
Total brain sampling will be performed using the brain
blowing technique devised by Veech et al (Biochem. J. 134,
1001, 1973). In order to sample regional samples of the
brain like the diencephalon where the appetite center is
located, the animals will be decapitated above liquid freom
maintained at -150 with liquid nitrogen. The brain will
be dissected in the frozen state. Alternatively, brains
will be coagulated by microwave irradiation of live animals,
Acetylcholine will be determined by radioenzymatic
assay as described by Goldberg and Mccaman (J. Neurochem.
20, 1, 1973). Acetylcholine synthesis will be measured in
vitro in brain slices and in nerve cell cultures;
slices or isolated cells will be incubated with 140-glucose
Brain
eserine and various concentrations of HC.
will be isolated by the method of Goldberg and Mccaman
14C-acetylcholine
before being counted.
In addition, the activities of citrate cleavage enzyme
and choline acetyl-transferase will be assayed in the brain
animals.
of rats chronically treated with HC as well as in control
4 .
Effect of hydroxycitrate on the enzymes of cholesterol
synthesis and cytoplasmic ketogenesis.
The following cytoplasmic enzymes will be
investigated :
acetoacetyl-CoA thiolase, HMG-COA
(continued)
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