TPM and Energy Balance, Picard et al.
channels (15). The fifth known property is an inhibitory
effect on carbonic anhydrase (CA), particularly CA-II and
CA-IV (16). Although the modulatory effect of TPM on
GABAA receptors differs from that of other AEDs (4), this
property would be expected to promote weight gain rather
than weight loss. Recently, evidence has been reported
suggesting that AEDs, which inhibit the activity of some
glutamatergic receptors, could promote weight loss (2). A
possible role for the glutamate N-methyl-D-aspartate recep-
tor in body weight control was suggested by a study in
which the N-methyl-D-aspartate receptor coagonist glycine
stimulated feeding behavior when administered into the
lateral hypothalamus of rats (17).
The observation that TPM may exert reducing effects on
body weight in humans prompted us to undertake a series of
experiments in the rat, with the objective of describing in
detail the components of energy balance that are affected by
the drug. To this end, a 2 ϫ 3 factorial experiment was
performed in which two cohorts of Zucker rats differing in
their phenotype (phenotype: lean, Fa/?; obese, fa/fa) were
each divided into three groups defined by the dose of TPM
administered (dose: TPM 0, vehicle; TPM 15, 15 mg/kg;
TPM 60, 60 mg/kg).
Institute (Raritan, NJ). The doses were selected based on
previous pharmacokinetic trials (4). One-third of the dose
was given in the morning, and the remaining two-thirds
were administered 2 hours before dark to obtain maximal
effects of TPM during the period of peak activity of
the animals. The doses of TPM were adjusted every other
day after the recording of body weight. Rats were treated
for 4 weeks.
Body Weight, Food Intake, and Body Gains in Energy,
Fat, and Protein
Throughout the study, body weight and the amount of
food ingested were monitored every other day. Food spilled
on the absorbent paper was carefully collected, allowed to
dry, and accounted for in the food-intake calculations. En-
ergy intake was calculated by multiplying cumulated in-
takes of food by the digestible energy (DE) content of the
diet. The DE was determined as being 95% of the gross
energy density of the diet. This determination was based on
previous studies (18,19), in which the energy content of the
feces was analyzed.
At the end of the experimental treatment, overnight-
fasted rats (from 7:00 AM to the time of killing [1:00 PM])
were anesthetized with an intraperitoneal injection of 0.4
mL/kg of a ketamine (20 mg/mL) and xylazine (2.5 mg/mL)
solution. The rats were killed between 1:00 PM and 3:00 PM.
The time of killing alternated among rats to balance the
effect of fasting duration among groups. Blood was har-
vested immediately thereafter by cardiac puncture and cen-
trifuged (1500 ϫ g, 15 minutes at 4 °C); the separated
plasma was stored at Ϫ70 °C until later biochemical mea-
surements. Carcasses were autoclaved at 125 kPa for 15
minutes. This procedure, which had been reported not to
affect energy yield (20), was used to soften hard tissues.
Once autoclaved, carcasses were homogenized in a volume
of water corresponding to two times their weight. The
homogenized carcasses were then freeze-dried pending the
determination of their energy and nitrogen contents. Carcass
energy content was determined by adiabatic bomb calorim-
etry, whereas carcass nitrogen was determined in 250- to
300-mg samples of dehydrated carcasses using the Kjeldahl
procedure. Carcass protein content was computed by mul-
tiplying the nitrogen content of the carcass by 6.25. The
energy as protein was subtracted from total carcass energy
to determine energy as nonprotein matter. Because carbo-
hydrate represents a negligible part of carcass total energy
(21), energy from nonprotein matter was assumed to be
essentially that of fat. Such an assumption tends to be
confirmed by studies in which energy, fat, and protein were
directly determined (22). Values of 5.62 and 9.39 kcal/g
were used for the calculation of the energy content of
protein and fat, respectively (21). Initial energy, fat, and
protein contents of the carcasses were estimated from the
live body weight of lean and obese rats with reference to a
Research Methods and Procedures
Animals and Treatments
Lean (Fa/?) and obese (fa/fa) female Zucker rats, aged 4
to 5 weeks, were purchased from the Canadian Breeding
Laboratories (St-Constant, Canada). Females were used in-
stead of males because the latter were not available at the
time this study was realized. Gender-dependent effects of
TPM were not expected a priori, as TPM had not been
systematically tested for its effects on body weight. All rats
were cared for and handled in accordance with the Canadian
Guide for the Care and Use of Laboratory Animals. Obese
and lean rats were age-matched at the beginning of the
study. Rats were individually housed in stainless cages
under controlled temperature (23 Ϯ 1 °C) and lighting
(light, 6:00 AM until 4:00 PM; dark, 4:00 PM until 6:00 AM).
The animals were allowed unrestricted access to food and
water. Throughout the study, rats were given a purified,
high-carbohydrate diet, which was composed of the follow-
ing (in g/100 g): 31.2 cornstarch, 31.2 DL -dextrose, 6.4 corn
oil, 20.0 casein, 0.3 DL -methionine, 1.0 vitamin mix (Tek-
lad no. 40060; Teklad, Madison, WI), 4.9 AIN-76 mineral
mix (ICN Biochemicals, Montre´al, Canada), and 5.0 fiber
(Alphacel; ICN Biochemicals). The energy content of the
diet consisted of 64.9% carbohydrate, 14.5% fat, and 20.6%
protein, and its density was 4.01 kcal/g. A week after their
arrival, both lean and obese rats were chronically treated
either with vehicle (dose 0) or TPM at two doses (15 and 60
mg/kg) given by gavage. TPM (RWJ-17021–000-DO) was
provided by the R.W. Johnson Pharmaceutical Research
OBESITY RESEARCH Vol. 8 No. 9 December 2000 657