This study examined the effects of different conjugated linoleic acid (CLA) isomers on body composition in adult male rats. Rats were fed diets containing either no CLA, the cis-9, trans-11 CLA isomer, the trans-10, cis-12 CLA isomer, or both isomers. Half the rats exercised daily via treadmill running. The study found that CLA intake, regardless of isomer or exercise, had no effect on food intake, body weight changes, or body chemical or anatomical composition in adult male rats. Physical activity also did not interact with CLA treatment to affect these outcomes.
This study examined the effects of different conjugated linoleic acid (CLA) isomers on body composition in adult male rats. Rats were fed diets containing either no CLA, the cis-9, trans-11 CLA isomer, the trans-10, cis-12 CLA isomer, or both isomers. Half the rats exercised daily via treadmill running. The study found that CLA intake, regardless of isomer or exercise, had no effect on food intake, body weight changes, or body chemical or anatomical composition in adult male rats. Physical activity also did not interact with CLA treatment to affect these outcomes.
This study examined the effects of different conjugated linoleic acid (CLA) isomers on body composition in adult male rats. Rats were fed diets containing either no CLA, the cis-9, trans-11 CLA isomer, the trans-10, cis-12 CLA isomer, or both isomers. Half the rats exercised daily via treadmill running. The study found that CLA intake, regardless of isomer or exercise, had no effect on food intake, body weight changes, or body chemical or anatomical composition in adult male rats. Physical activity also did not interact with CLA treatment to affect these outcomes.
Cis-9, Trans-11 and Trans-10, Cis-12 Conjugated Linoleic Acid
Isomers Do Not Modify Body Composition in Adult Sedentary or Exercised Rats1 Philippe Patureau Mirand 2 , Marie-Agns Arnal-Bagnard, Laurent Mosoni, Yannick Faulconnier * ,Jean-Michel Chardigny and Yves Chilliard *
Unit Nutrition et Mtabolisme Protique and; * Unit Recherches sur les Herbivores, Centre Inra de Clermont-Ferrand-Theix, 63122 Theix, France and; Unit Nutrition Lipidique, Centre Inra de Dijon, 21065 Dijon, France 2 To whom correspondence should be addressed. E-mail: patureau@clermont.inra.fr.
ABSTRACT TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED
Dietary CLA isomers were shown to reduce adipose tissues in
growing animals, mainly in mice, but their effects in adult
animals remain unclear. This study was conducted to determine
whether these effects depend on the isomer fed, on physical
activity, or on the initial level of body fat. Male Wistar rats
(4 mo old) were fed for 6 wk diets containing either no CLA,
the cis-9, trans-11 CLA isomer (10 g/kg), the trans-10, cis-12
CLA isomer (10 g/kg), or both isomers (10 g/kg each). Half of
the rats were assigned to exercise by treadmill running (1 h/d,
22 m/min). The initial body fat level was normal (12.7%) in
a first trial, and high (18.9%) in a second trial. Chemical
and anatomical body compositions were determined by chemical
analysis and organ dissection. In both trials, the CLA diets,
whatever the isomer, had no effect on food intake and body weight
changes, on body chemical composition (fat, protein and water
contents or gains), or on the body anatomical composition (weights
or gains in epididymal and perirenal adipose tissues, in liver
and in 4 muscles). There was no interaction between CLA treatment
and physical activity. In conclusion, adult male rats do not
appear to be responsive to the fat-to-lean partitioning effect
of CLA described in growing rats. This was not affected by exercise
or initial body fat level.
KEY WORDS: CLA isomers exercise body composition rats The consequences of feeding conjugated linoleic acid isomers
(CLA) on whole-body composition appear to be unclear, probably
because they depend on numerous factors related either to the
subjects (genotype, age, gender, physiologic and nutritional
status), or to the treatment (isomers, level of feeding, duration
of treatment). In mice, since the first experiment by Park et
al. in 1997 (1), consistent data indicate that dietary CLA decreases
adiposity in growing animals (26) and in adults (710),
both male and female (25). CLA feeding was described
as slightly increasing lean body mass in growing mice (11).
The fat-reducing effect was associated with the trans-10, cis-12
isomer of CLA (5,12), whereas the cis-9, trans-11 had a growth- promoting
effect (10). Less consistent effects were described in rats, hamsters, pigs,
and humans. In growing female rats, CLA significantly decreased
adipose tissues (1315). In growing male rats, the reported
effects were less constant, i.e., a significant decrease was
reported for retroperitoneal pad (15,16) or perirenal pad (1719)
but not for epididymal pad (1618). CLA feeding did not
affect fat pad weights in obese Zucker male rats (16) or in
growing male rats (14), but soleus weight was increased in the
latter study (14). In growing hamsters, a CLA isomer mixture
had a slight fat-reducing effect without effect on protein mass
(20); it could also prevent an increase in adiposity, whereas
the cis-9, trans-11 isomer increased it (21). In growing-finishing
pigs, CLA intake reduced adiposity linearly (22) or quadratically
(23,24). In this case, the maximum effect was obtained withdifferent doses according to age: 5 g/kg food during the first
4 wk of the growing-finishing period and 2.5 g during the last
4 wk. This may explain why other studies did not detect any
effect of CLA feeding on body composition (25,26). In humans,
conflicting results were described. In a recent review (27),
7 studies conducted among overweight subjects were compiled.
In 3 studies, CLA feeding reduced the body fat percentage and
the sagittal abdominal diameter, and it had no significant effect
in the other 4 studies. Recently, CLA effects on weight regain
and body composition were studied in overweight subjects after
weight loss (28). CLA did not prevent weight regain but increased
fat-free mass regain. In 2 other studies, the effects of CLA
were tested in exercising healthy humans of normal body weight,
and conflicting results were described. CLA supplementation
reduced the body fat percentage in 1 experiment (29) but had
no significant effect on total body mass, fat-free mass, fat
mass, percentage of body fat, bone mass, or strength in another
experiment (30). Thus, the specific effects of feeding CLA isomers on body composition
in adults and whether these effects might depend on initial
fatness or onphysical training remain unclear. The present
study was designed to determine the effects of CLA intake (cis-9,
trans-11 and/or trans-10, cis-12 isomers) on body chemical and
anatomical compositions in adult rats and to establish whether
physical training or initial body fat level could interact with
CLA feeding. Two trials with different initial body fat levels
(normal or high) were performed. Half of the animals in each
trial were assigned to treadmill exercise to test the possibility
that exercise would affect the efficiency of CLA treatments.
MATERIALS AND METHODS TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED
Animals, diets, and exercise. In each trial, male Wistar rats (n = 55) were purchased from
Iffa-Credo/Charles River. In trial 1, they were 15-wk-old rats
and had been fed a 20% protein diet since weaning (lean rats).
In trial 2, the rats were 17 wk old and the protein content
of the diet they were fed since weaning was 17.5% (fat rats).
They were maintained in individual wire-bottom cages at 21C
with a 12-h light:dark cycle (lights on at 2000 h) and free
access to water. The adaptation period lasted 7 d. In each trial,
a group of 7 rats (group 0) was killed and dissected at the
end of the adaptation period to verify the body composition
at the beginning of the experimental period. The 48 remaining rats were divided into 2 groups. The 24 trained
rats were exercised by treadmill running. The rats were progressively
adapted during wk 1 to run for an hour at 22 m/min (i.e., no
>50% VO2 max). They were exercised for 6 wk, 6 d/wk during
the dark period (at 1500 h). The other group (n = 24) was not
assigned to exercise (sedentary rats). Four diets were tested: the control diet (Control), the control
diet with 10 g/kg cis-9, trans-11 CLA (c9,t11), the control
diet with 10 g/kg trans-10, cis-12 CLA (t10,c12), and the control
diet with the same amounts (10 g/kg) of each isomer (Mixture).
The 4 diets had the same basal composition (g/kg): casein 180,
cornstarch 430, sucrose 210, cellulose 20, mineral mixture 50,
vitamin mixture 10 (31), oil mixture 100. They differed in the
composition of the oil mixture (Table 1). The CLA isomers, provided
as triglycerides (Natural Lipids), were substituted for high-oleic
sunflower oil. The diets were fed in a semiliquid form to allow
better control of food intake.
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TABLE 1 Composition of the oil mixtures fed to male Wistar rats for 6 wk Each diet was consumed ad libitum for 6 wk by 6 rats in each
of the sedentary and exercised groups. During this period, daily
food intake was measured 5 d/wk. The rats were weighed 3 times/wk.
All procedures were performed according to current legislation
on animal experimentation in France. Whole-body composition. General anesthesia was induced by i.p. injection of pentobarbital
(left and right gastrocnemius, extensor digitorum longus, tibialis
anterior, and soleus) and 2 adipose tissues (perirenal and epididymal)
were quickly removed. All organs were weighed; the digestive
tract was weighed twice, before and after it was emptied. The
remaining carcass was frozen and stored at 20C until
determination of its chemical composition. The frozen carcass
of each rat was pulverized with a grinder (Robot-Coupe) to obtain
a homogenous frozen powder. Two representative samples of each
carcass were freeze-dried. Residual water, protein, lipids,
and minerals were determined in these samples. Residual water
was measured by desiccation at 103C for 48 h. The nitrogencontent was measured by the Kjeldahl method. The protein content
was calculated as the nitrogen content x 6.25. Total lipids
were determined by the method of Folch (32). Minerals were measured
as the ash content after incineration at 500C for 6 h.
Lipids and water in the adipose tissues sampled were added to
the value for the carcass. The biochemical composition of the
empty body was calculated from the values obtained for carcass
and adipose tissues, assuming that the mean composition of liver,
blood collected, intestines, and the 4 muscles was not different
from whole-body composition. This could not affect the global
composition because this group of organs and tissues represented
<6% of whole-body weight. Calculations and statistical analysis. The empty body weight was determined as the difference between
the whole-body weight and that of the digestive contents. The
body composition was expressed/100 g empty body weight. The
variations in organ weights during the experimental period were
calculated from the difference in organ weights measured at
the end of the trial and estimated at d 0. Organ weight at d0 was estimated in all rats by multiplying their respective
body weight by the mean organ weight per unit of body weight
measured directly in the 7 rats killed on d 0. The biochemical
composition of weight gain was calculated from the difference
in the amount of constituents measured at the end of the experimental
period and that estimated at d 0. Whole-body biochemical composition
at d 0 was estimated in all of the rats by multiplying their
mean body weight by the empty body composition in the 7 rats
killed on d 0. A Students t test was performed to determinewhether variation in body composition or organ weights during
the trial differed from zero or to compare the initial body
composition in lean and fat rats. In each trial, data were subjected
to a 2-factor ANOVA to detect the effects of diets, exercise,
and their interaction. When the effects of the diets were significant
(P < 0.05), differences between diets were determined using
Fishers protected multiple comparison test. Means of
main effects (diet and exercise) and pooled SEM are reported.
Differences are considered significant when P < 0.05. The
StatView statistical software package (version 5 SAS Institute)
was used for all of the statistical analyses.
RESULTS TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED
I nitial body composition. The fat rats (trial 2), which had an initial body lipid content
higher (+48%) than that of the lean rats (trial 1), also had
lower water, protein and mineral contents (Table 2). These differences
were confirmed by anatomical measurements. The relative weights
(expressed as a percentage of empty body weight) of perirenaladipose tissue and liver were higher in the fat group than in
the lean group and soleus muscle relative weight was lower.
However, epididymal adipose tissue weights did not differ.
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TABLE 2 Initial whole-body composition of male Wistar rats fed diets with or without various CLA isomers for 6 wk 1
Food intake and growth rates. Two rats died for undetermined reasons in trial 2. One was in
the exercised group and was fed the control diet; the other
was in the sedentary group and was fed diet t10,c12 (with trans- 10,
cis-12 isomer). They were withdrawn from the study and not replaced. In both trials, food intake, weight gains, and food efficiency
were not affected by diets but they were significantly lower
in the exercised rats than in the sedentary rats (Table 3).
There was no significant interaction between diets and exercise
for dry matter intake, weight gain, or food efficiency whatever
the initial body fat level.
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TABLE 3 Effects of conjugated linoleic acid and exercise on food intake, weight gain, and food efficiency in lean and fat rats fed diets with or without various CLA isomers for 6 wk 1
Body composition. In both trials, the diets had no influence on water, protein,
and lipid contents in whole body (Table 4). Exercised rats hada lower lipid content and higher water and mineral contents
than the sedentary rats. There was no significant interaction
between diet and exercise on body chemical composition in the
lean and the fat rats.
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TABLE 4 Effects of conjugated linoleic acid and exercise on whole-body biochemical composition in lean and fat rats fed diets with or without various CLA isomers for 6 wk 1
In the fat rats, as in the lean rats, the diets did not affect
the relative weights of the 2 adipose tissues studied, the liver,
or the 4 muscles studied (Table 5). Exercise induced higher
relative muscle weights (except in the soleus of the fat rats).
On the contrary, the relative weights of perirenal and epididymal
adipose tissues were lower in exercised rats than in sedentary
rats. Exercise did not affect relative liver weights. There
was no significant interaction between diet and exercise on
organ weights whatever the initial body fat level.
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window] [in a new window]
Weight gain and changes in organ weight during the experimental periods. In the lean and the fat groups, diets did not affect the biochemical
composition of weight gains (Table 6) except for minerals. Mineral
gains during the experiment were significantly lower in rats
fed diet c9,t11 than in those fed the control diet. Exercised
rats had lower lipid, water, and protein gains than sedentary
rats. Changes in organ weights during the experimental period
were not affected by the diets whatever the initial body fat
level (Table 7). In both trials, exercise lowered the empty
body weight gain as well as the increase in the weight of perirenal
and epididymal adipose tissues, and of liver (nonsignificant
in fat rats, P = 0.18) but it did not affect the weight changes
in gastrocnemius, extensor digitorum longus, and tibialis anterior
muscles.
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TABLE 6 Effects of conjugated linoleic acid and exercise on the biochemical composition of body weight gain in lean and fat rats fed diets with or without various CLA isomers for 6 wk 1
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TABLE 7 Effects of conjugated linoleic acid and exercise on the variations of empty body and organ weights in lean and fat rats fed diets with or without various CLA isomers for 6 wk 1
DISCUSSION TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED
In these experiments, feeding high-fat diets (100 g/kg) with
10 g/kg of cis-9, trans- 11, trans-10, cis-12, or both CLA isomers
for 6 wk to 4-mo-old rats as triacylglycerols had only minor
effects on their body weight changes and on their body composition
tested at 2 initial body fat levels and 2 levels of physical
activity. To our knowledge, this is the first time that the
effects of CLA on body composition obtained in rats > 13
wk old are reported. Moreover, the consequences of the initial
fat status or exercise training interaction were tested. The initial fat status was either normal or high for 16- to
18-wk-old Wistar rats (33). This difference was not the only
consequence of the slight age difference (2 wk) between the
2 groups. Indeed the body fat content of the 22-wk-old lean
rats (at the end of the experimental period) did not equal that
of the 18-wk-old fat rats (at the beginning of this period)
despite the high-fat diet they were fed for 6 wk. This allowed
us to test the effects of CLA feeding and exercise in the same
strain of rats at 2 levels of body fat status due primarily
to the diets fed after weaning. Physicalexercise in this study
(treadmill running at 22 m/min for 1 h) can be considered to
be low-intensity endurance training, <55% VO2 max for malerats (34,35). It decreased food intake and weight gain and induced
a decrease in body lipid content and adipose tissue weights.
Consequently, the lipid and adipose tissue gains were lower
in exercised rats than in sedentary rats. On the contrary, body
water concentrations and also muscle relative weights were increased
by exercise. Furthermore, it can be calculated that the amount
of dietary protein not used to increase whole-body protein content
was slightly higher (P = 0.05) in exercised rats than in sedentary
rats (2.96 0.4 and 3.06 0.4 g/d, respectively).
This demonstrates that exercise had no sparing effect on protein
in this context of generous protein feeding (165 mg/g food dry
matter) and restricted energy (10% compared with sedentary
rats). All of these results are consistent with data from the
literature on the consequences of endurance training in rats
(3638). The effects of CLA supplementation were less marked. Food intake
was not affected by CLA feeding. Depressive effects were described
mainly in growing mice fed diets supplemented with 510
g CLA isomer mixtures/kg (1,2) but not in growing rats whose
diets were supplemented with 5 g CLA isomer mixture/kg (13,14,16,19).
Body weight changes were not affected by CLA feeding, which
agrees with most results obtained in growing rats consuming
feed ad libitum (13,14,19). Similarly, no effect of CLA supplementation
on body chemical composition could be detected, in keeping with
the few studies in which this composition was determined in
rats (20,39). CLA supplementation had no effect on anatomicalcomposition either. This is consistent with the literature concerning
rats for liver (13,15,16,18,19,39,40), epididymal adipose tissue
(14,1619), and gastrocnemius muscle (1316,40).
For soleus muscle, it was reported that the CLA mixture induced
an increase in muscle weight in male rats but not in females
(14); however, in other studies (13,15,41) no effect was reported,
as is the case in the present experiment. It is more surprisingthat no effect of CLA isomer feeding was detected on the weight
of perirenal adipose tissue because it was reported to be decreased
in the 4 studies in which it was measured in rats (17 19,42).
Furthermore a lowering effect of CLA feeding was usually described
in rats for most other adipose tissues (inguinal, retroperitoneal,
parametrial), except for epididymal. In summary, the lack of
an effect of CLA on food intake, growth, and CLA-non responsive
parameters of body composition (lean body mass, muscles, liver,
epididymal adipose tissue) confirms in adult male rats what
was already observed in young rats. On the contrary, the decrease
in perirenal adipose tissue induced by CLA feeding in young
male rats was not found in the present study with adult male
rats. It is still unknown whether the same age-related discrepancy
exists for other CLA responsive adipose tissues such as inguinal
or retroperitoneal tissues. However, it was shown in 11-wk-old
female rats (14) and in 26- to 30-wk-old mice (9) that CLA feeding
reduced fat pads, as in younger animals. This indicates that
adult male mice or female rats are more responsive than adultrats. Concerning potential specific effects of each isomer or
of an interaction between both isomers, none could be detected
on body composition. This contrasts with data from mice (5,12,43),
from obese rats (41), and from hamsters (44) that demonstrated
a specific fat-lowering effect of the trans-10, cis-12 isomer.
The absence of CLA effect at both initial body fat levels in
these male Wistar rats is also different from what was reported
in mice and in female Zucker rats. Indeed, a CLA mixture reduced
adipose tissue similarly in lean and fat mice (9) and in the
lean line of rats, but increased it in the obese line (16).
This discrepancy could result from the species difference, from
the very high level of body fat in obese Zucker rats, and/or
from the specific cause of obesity in these rats. A synergistic
effect with exercise was also hypothesized because exercise
increases energy losses, thus limiting energy available for
fat deposition and fat mass gain. Furthermore, CLA supplementationproved to be effective in restricted subjects such as growing
rats (39) and adult dogs (45). This may contribute to a part
of its fat-lowering effect in exercising men (29). However,
in the present study, CLA feeding had the same consequences
on body composition in exercised and in sedentary rats. Globally,
the parameters related to food intake, growth rate, and chemical
and anatomical composition that were not modified by CLA feeding
in young male rats were not affected in adults either. However,
4 main discrepancies were found between this study and data
in the literature, including perirenal adipose tissue weight,
specific effect of cis-10, trans-12 isomer on body composition,
effect of initial fat status, and exercise. It seems unlikely
that these discrepancies can be explained by the fact that CLA
isomers were fed as triacylglycerols rather than FFA because
it was shown in mice that both forms have similar effects on
body composition (46). These discrepancies are likely the consequence
of age or result from species differences. In conclusion, this experiment confirms the effects of physical
exercise on body composition changes that were already known,
but it has not been possible to detect any effect of the 2 major
CLA isomers on body fat and body fat-free masses in lean or
fat adult rats. The comparison with growing rats suggests that
adipose tissues (epididymal excepted) in adult male rats could
be less responsive to the fat-reducing effect of the CLA mixture
(or of the trans-10, cis-12 isomer). However, this does not
mean that CLA isomers had no effect on lipid, protein, or energy
metabolisms in adult rats because metabolic rates can be altered
without any consequence on body composition; such aspects deserve
further studies to elucidate the real effect of these compounds
on major metabolic pathways.
ACKNOWLEDGMENTS
Christine Cubizolles is specially acknowledged for animal facilities,
Christian Lafarge for animal management, and Danielle Bonin
and Hlne Lafarge for help in the literature
search.
FOOTNOTES
1 Conducted with financial support from the Commission of the
European Communities specific RTD programme "Quality of Life
and Management of Living Resources" QLK1- CT9900076 "Conjugated
Linoleic Acid (CLA) in functional food: a potential benefit
for overweight middle-aged Europeans (FunCLA)." It does not
necessarily reflect its view and in no way anticipates the Commissions
future policy in this area.
Manuscript received 8 April 2004. Initial review completed 28 April 2004. Revision accepted 7 June 2004.
Increased Consumption of Dairy Foods and Protein During Diet - and Exercise-Induced Weight Loss Promotes Fat Mass Loss and Lean Mass Gain in Overweight and Obese Premenopausal Women