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Department of General and Environmental Physiology, University of Naples "FEDERICO II", Italy I-80134
2To whom correspondence should be addressed at Dipartimento di Fisiologia Generale ed Ambientale, Via Mezzocannone 8, I-80134, Napoli, Italy. Fax: +39-081-5426868. E-mail: susiossa{at}unina.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • energy balance measurements • resting metabolic rate • leptin • developing rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, Iossa et al. 1997
,
Lionetti et al. 1996a
, Lionetti et al. 1996b
, Liverini et al. 1994
,
Liverini et al. 1996
). On the other hand,
both humans and rodents develop a spontaneous obesity as they grow
older (Barzilai and Rossetti 1995
, Masoro 1980
, Newby et al. 1990
). Thus, in the older
rats, some regulatory mechanism which allows maintainance of body
weight can fail. In an attempt to gain further insights into the energetic aspects of the tendency to accumulate fat typical of rats as they grow old, we evaluated the relative importance of changes in energy intake and expenditure in developing rats. Since, to our knowledge, a complete study on the energy utilization in maturing Wistar rats has not been done, we carried out measurements of energy balance and changes in body energy compartments in young rats in which body fat is quite low. These animals were then followed until adult age when the obesity propensity starts to manifest. In addition, to have more information about the role of energy metabolism in body weight control, resting metabolic rate (RMR)3 was measured after 16 h of food deprivation by using indirect calorimetry. Variations in serum free triiodothyronine (FT3), leptin, and free fatty acids (FFA) were also checked.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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At the start of the study, 28 rats (aged 25 d) were divided into seven groups, each composed of four rats, with similar mean body weight. One group was killed for the estimation of energy content and body composition of 25-d-old rats. The other six groups were killed at 40, 55, 90, 120, 150, and 180 d of age. At each time of death, the killed animals had a mean and range of body weight equal to that of the other groups of animals. Therefore, in our calculations of energy gain, we assumed that the surviving animals contained the same proportions of fat, protein, and water in their carcasses as those in the killed animals.
The day before the killing, the rats of each group were food-deprived for 16 h (from about 1700 h), and the following morning they were used for RMR determination. The oxygen consumption was measured in an open-circuit oxygen consumption system (O2-ECO; Columbus Instruments, Columbus, OH), designed to monitor oxygen consumption in small animals. The instrument was calibrated with room air before and after each measurement. Although most rats quieted down after about 30 min in the chamber, all rats were allowed to adapt to the experimental environment for a minimum of 60 min before beginning the measurements. RMR in food-deprived rats was measured in a chamber at 24°C over a period of at least 10 min during which the rat remained quiet.
Measurement of body composition.
After RMR measurements, the rats were anesthesized with chloral
hydrate (40 mg/100 g body wt), and blood was collected via the inferior
caval vein. Then, the carcasses were weighed, autoclaved for 90 min,
chopped into small pieces, thoroughly mixed, and finally homogenized
with water (volumes equal to twice the carcass weight) in a Polytron
homogenizer. Aliquots of the homogenate were analyzed for lipid and
water content. Lipid content was determined gravimetrically after
extraction in chloroform–methanol and evaporation to constant weight
by a rotating evaporator (Heidolh, Germany) by the method of
Folch et al. (1957)
. The energy as lipid was calculated
from the lipid content by using the coefficient of 39.2 kJ/g for the
energy content of lipid. Water content was determined by the difference
in weight of the homogenate before and after drying at 70°C in a
vacuum oven. Then, small pellets (about 200 mg) of the dried homogenate
were made, and the body energy content was measured with a bomb
calorimeter (Parr adiabatic calorimeter; Parr Instruments Co., Moline,
IL). The energy as protein was calculated from a general formula
relating lipid-derived energy, total energy value of the carcass,
and protein-derived energy. The formula is based on the fact that
lipid and protein are the only energy-yielding components of the
carcass with a negligible (<1%) contribution from carbohydrates
(Dulloo et al. 1990
). Protein content was obtained by
using the value of 22.4 kJ/g for the energy content of protein.
Energy balance measurements.
Body weights and food intakes were monitored daily to allow calculations of body weight gain and gross energy intake. The feces and spilled food were also collected daily, dried, and ground to a powder before determining their energy content with the bomb calorimeter. The gross energy content of the stock diet was also determined by bomb calorimetry.
Digestible energy intake was obtained by subtracting the energy
measured in the feces and spilled food from the gross energy intake as
measured from daily food consumption. Metabolizable energy (ME) intake
was expressed as digestible energy intake x 0.96 (Barr and McCracken 1984
). The gain in energy was obtained from the
difference between the final body energy content at the end of each
period and the energy content of the animals killed at the end of the
previous period. Energy expenditure was calculated from the difference
between ME intake and energy gain.
Serum metabolites.
Serum samples were stored at -20°C until the time of measurement. Serum FT3 and leptin levels were measured using commercial radioimmunoassay kits (Coat A Count; Diagnostic Products Corporation, Los Angeles, CA for FT3, and Mediagnost, Germany for leptin). Inter- and intraassay coefficients of variation were 7 and 4% for T3 assay and 6 and 9% for leptin assay. Serum FFA levels were measured by colorimetric enzymatic method using a commercial kit (Boehringer Mannheim, Italy).
Statistical analysis.
Data are given as means ± SEM of four different rats. Statistical analyses were performed by one-way analysis of variance (ANOVA). Post-hoc comparison between group pairs was made with the Tukey test after ANOVA had established significant differences among groups. Regression analyses were also performed. Probability values less than 0.05 were considered to indicate a significant difference. All analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Body weight and body energy/g body wt of developing rats from 25 to
180 d of age are reported in Figure 1
A. Before the sexual maturity, an initial rapid growth period occurred;
then, the gain in body mass slowed. Body energy/g body wt significantly
increased between 40 and 55 d of age; a further increase occurred
between 120 and 150 d of age. Body water decreased with age,
from about 73% of body weight in the youngest rats to about 60%
in the oldest rats (Fig. 1B)
. The percentage of the body protein did
not significantly vary with age, although there was a tendency
(P = 0.07) to decrease in rats of 150 and 180 d of age. Percent body lipid significantly increased in developing
rats, from about 5% in 25-d-old rats to about 17% in 180-d-old rats
(Fig. 1B)
.
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Both daily ME intake and energy expenditure expressed per
metabolic body size (kg0.75) significantly decreased in
developing rats up to 90 d of age and remained constant
thereafter (Fig. 2
A). RMR expressed per g body protein exhibited the highest value in
25-d-old rats. Then, RMR significantly decreased, reaching a constant
value from 55 to 180 d of age (Fig. 2A)
.
|
. The total cost of storage was determined taking into
account that the energy loss in storing 1 kJ of protein is 1.25 kJ
(Pullar and Webster 1977
), while the corresponding
energy cost for fat deposition is 0.36 kJ/kJ for diets with a high
percentage of carbohydrates (Pullar and Webster 1977
),
such as the diet used here. The percentage of ME intake used for
storage of protein and lipid significantly decreased with age.
Expressed as a percentage of ME intake, values obtained for energy
expenditure excluding the total cost of storage, corrected energy
expenditure, significantly increased in developing rats up to 120 d of age and remained unchanged thereafter. The percentage of energy
intake stored as protein significantly decreased with age, while the
percentage stored as fat reached the lowest value at 120 d of age
(Fig. 2B)
.
The percentage of ingested protein which was stored as carcass energy
significantly decreased, especially after 90 d of age, while the
percentage of ingested lipid which was stored reached the lowest value
at 120 d of age and then increased in 150 and 180-d-old rats (Fig. 2C)
.
Serum metabolites.
Serum FFA significantly increased only in 180-d-old rats
(Fig. 3
A). Serum FT3 showed a peak at 55 d of age and did not
significantly vary thereafter. Serum leptin levels significantly
increased in developing rats, reaching a constant value at 150 and
180 d of age. Leptin levels were strongly correlated with body fat
mass (Fig. 3B)
and daily ME intake/kg0.75 (Fig. 3C)
.
However, regression lines for the correlation between leptin levels and
ME intake showed significantly different slopes during the period
25–90 and 90–180 d.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, coupled with a
significant storage of body protein (Fig. 2)
, was followed by a lower
rate of growth, with protein deposition that approached zero (Fig. 2)
.
However, ME intake remained about 10% higher than the energy needs for
maintenance (Fig. 2)
. This excess of energy was entirely stored as fat
(Fig. 2)
. Therefore, the ingested proteins, which in rapidly growing
rats were partly stored in the body and partly used to replace stores
(Fig. 2)
, were at this time used only to support protein turnover. In
agreement, we can calculate that protein oxidation contribution to
total energy expenditure goes from about 20% in 40-d-old rats to about
30% in 180-d-old rats.
Up to 55 d of age, body energy gain as lipid and lipid intake was
almost equivalent, while starting from 55 d of age lipid gain
represented only a part of lipid intake (Fig. 2)
. It follows that
an increase in fat utilization for metabolic needs takes place at this
stage, although serum FFA levels increased only in 180-d-old rats (Fig. 3)
. Therefore, seemingly due to reduced energy intake from 55 d of
age, carbohydrate and protein oxidation was not sufficient to meet body
energy needs (Fig. 2)
.
The constant reduction in energy intake during development (Fig. 2)
may
be due to the increased lipid content of developing rats (Fig. 1)
. In
fact, it was recently proposed that the levels of some molecules, like
leptin, which are related to the adiposity of the body (Saladin et al. 1995
), could act in the hypothalamus to reduce food
intake (Schwartz et al. 1996
). Measuring serum leptin
levels, which were significantly increased in developing rats (Fig. 3)
,
tested this hypothesis. Serum leptin levels correlated significantly
with body fat mass and with daily ME intake/kg0.75 (Fig. 3)
. Interestingly, in the last correlation, the slope of the regression
line obtained for the period 90–180 d (-31.9 ± 7.4) was
significantly (P = 0.0068) higher than that obtained
for the period 25–90 d (-248.7 ± 10.2). This result suggests
that from 90 d of age onward a progressive reduction in the
responsiveness of the rat to the leptin signal with age takes place. Up
to now, increased leptin resistance was only found in old rats
(Li et al. 1997
, Qian et al. 1998
); our
results suggest for the first time the possibility that an early onset
of leptin resistance in developing rats is responsible for
age-associated obesity.
The possibility that the reduction in daily energy expenditure, which
occurs in developing rats, could be due to a decrease in basal
metabolism has been a matter of debate (McCarter & Palmer 1992
). However, to our knowledge, this question has not yet
been addressed. To supply such information, we measured RMR in
food-deprived rats at different ages (Fig. 2)
. RMR measured in this
condition involves the energy cost of sustaining the body's vital
functions, but not the thermic effect of food and locomotor activity,
which are the other components of daily energy expenditure
(Danforth 1992
). Our results show that RMR significantly
decreased from 25 to 55 d of age and remained constant thereafter
(Fig. 2)
. On the other hand, daily energy expenditure decreased in rats
with increasing age, reaching the lowest value in 90-d-old rats (Fig. 2)
. Thus, it can be suggested that the reduction in daily energy
expenditure found in growing rats is achieved by a decrease in the
thermic effect of food and/or locomotor activity. It should be noted
that serum FT3 levels correlated with RMR values between 60
and 180 d of age (r2 = 0.57,
P < 0.05), in agreement with the hypothesis that
T3 is the major hormonal determinant of RMR (Freake and Oppenheimer 1995
). On the other hand, the discrepancy
between RMR and serum FT3 levels in the younger rats could
be partly due to higher thermoregulatory needs of small animals at
24°C.
In conclusion, in the present work we found that the inability of adult rats to maintain a stable body weight cannot be attributed to a decrease in resting metabolism but rather to an early insensitivity to the satiating effect of leptin.
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FOOTNOTES |
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3 Abbreviations used: ANOVA, analysis of variance;
FFA, free fatty acid; FT3, free triiodothyronine; ME,
metabolizable energy; RMR, resting metabolic rate.
Manuscript received August 7, 1998. Initial review completed November 23, 1998. Revision accepted May 10, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, g; kJ/g, • and
(B) Body composition at different ages in Wistar rats. Each
point represents mean ± SEM, n = 4. *P
< 0.05 compared to the preceding age (not reported for body
weight values).
