Nutrient Metabolism

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

sodium
(Sanofi). Liver, digestive tract, heart, 4 skeletal muscles

(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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TABLE 5 Effects of conjugated linoleic acid and exercise on organ weights in lean and fat rats fed diets
with or without various CLA isomers for 6 wk
1

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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.