Vol. 282, Issue 6, R1789-R1797, June 2002
trans-10, cis-12, but not cis-9,
trans-11 CLA isomer, inhibits brown adipocyte
thermogenic capacity
Enrique
Rodríguez,
Joan
Ribot, and
Andreu
Palou
Departament de Biologia Fonamental i Ciències de la
Salut, Universitat de les Illes Balears, Cra Valldemossa, 07071 Palma de Mallorca, Spain
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ABSTRACT |
Conjugated linoleic acid (CLA) is
reported to have health benefits, including reduction of body fat.
Previous studies have shown that brown adipose tissue (BAT) is
particularly sensitive to CLA-supplemented diet feeding. Most of them
use mixtures containing several CLA isomers, mainly cis-9,
trans-11 and trans-10, cis-12 in equal
concentration. Our aim was to characterize the separate effects of both
CLA isomers on thermogenic capacity in cultured brown adipocytes. The
CLA isomers showed opposite effects. Hence, on the one hand,
trans-10, cis-12 inhibited uncoupling protein (UCP) 1 induction by norepinephrine (NE) and produced a decrease in
leptin mRNA levels. These effects were associated with a blockage of
CCAAT-enhancer-binding protein-
and peroxisome
proliferator-activated receptor-
2 mRNA expression. On
the other hand, cis-9, trans-11 enhanced the UCP1
elicited by NE, an effect reported earlier for polyunsaturated fatty
acids and also observed here for linoleic acid. These findings could
explain, at least in part, the effects observed in vivo when feeding a
CLA mixture supplemented diet as a result of the combined action of CLA
isomers (reduction of adipogenesis and defective BAT thermogenesis that
could be through trans-10, cis-12 and enhanced UCP1
thermogenic capacity through cis-9, trans-11).
brown adipose tissue; uncoupling protein; fatty acid; leptin; adipogenesis; conjugated linoleic acid
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INTRODUCTION |
BROWN ADIPOSE TISSUE
(BAT) plays an important role in energy efficiency and body weight
control in small mammals because it is the main mediator of adaptive
thermogenesis (13, 25, 30). This largely depends on the
activity of the uncoupling protein (UCP) 1, a brown adipocyte-specific
inner mitochondrial membrane protein that allows the dissipation as
heat of the proton gradient generated by the oxidation of
nutrients (mainly fatty acids; see Ref. 29). Other
putative, UCP1-like uncoupling proteins, namely UCP2 and UCP3, have
been identified as new potential molecular targets for the regulation
of energy efficiency (4, 12), although this has not been
confirmed. These proteins are expressed in BAT and other tissues,
including those of humans (36).
The physiological regulation of adaptive thermogenesis by exogenous
factors depends primarily on stimulation of the sympathetic nervous
system, which densely innervates BAT (16). Release of norepinephrine (NE) plays a major role in brown adipocytes by stimulating cell proliferation and mitochondriogenesis and causing an
increase in UCP1 levels (8). These effects are mainly
mediated by the 
-adrenergic receptors, particularly important the
3-adrenergic receptor (3-AR), which has
been shown to mediate the stimulatory action on UCP1 synthesis and
activity in mature adipocytes (34, 49). In addition, UCP
gene expression is also regulated by other hormones and nutrients, such
as triiodothyronine, insulin, leptin, retinoic acid, and fatty acids
(36).
The acquisition and maintenance of the mature adipocyte function,
expressing the genes that control lipid metabolism and thermogenesis, are under the control of several transcription factors (15, 30,
39). These include members of the peroxisome
proliferator-activated receptor family (PPAR), a lipid-activated
subgroup of the nuclear hormone receptors, the CCAAT-enhancer-binding
proteins (C/EBP), and the adipocyte differentiation and determination
factor 1 (ADD1), a member of the sterol regulatory element-binding
proteins (39).
Conjugated linoleic acids (CLA) are a naturally occurring group of
positional and geometric isomers of linoleic acid (LA) formed by rumen
bacteria (9). The major dietary sources of CLA are beef
and dairy products (24). CLA consumption has been shown to
have a variety of health benefits as follows: protecting tissues from
carcinogenesis (19), reducing the development of atherosclerosis (23), stimulating the immune system
(26), and preventing diabetes (17) and
obesity (31, 46). CLA have been shown to affect body fat
content and energy metabolism in mammals (31, 44, 46),
including humans (2), by several mechanisms as follows:
reduced energy intake, increased energy expenditure, and an attenuation
of adipocyte cellularity and mass; CLA reduce adipogenesis, induce
apoptosis, and promote adipocyte delipidiation coupled with
enhanced fatty acid oxidation in both muscle cells and adipocytes
(6, 11, 27, 31, 32, 42, 44). It should be remarked that
many physiological effects of CLA in animal models and cell cultures
reported to date have been produced using mixtures of CLA isomers
(6, 10, 11, 32). However, there is recent evidence
suggesting that CLA-associated body composition changes and adipocyte
metabolism, in rodents and humans, result mainly from feeding the
trans-10, cis-12 CLA isomer (7, 11, 14, 32).
Previous studies have shown that BAT is particularly sensitive to
CLA-supplemented diet feeding, which causes BAT atrophy with defective
UCP1-dependent thermogenesis (44). Other studies reported
no CLA-associated changes in BAT weight or in UCP1 expression (40, 45). They used a CLA preparation containing several
CLA isomers (mainly the cis-9, trans-11 CLA and
trans-10, cis-12 CLA) present in similar amounts
(40, 44, 45) or a cis-9, trans-11 CLA-enriched diet (40). Hence, it is possible that either,
or both, of these isomers could be responsible for defective BAT thermogenesis induction. The aim of the present study was to examine the effect of both CLA isomers, separately or in combination, on
thermogenic capacity. Brown preadipocytes grown and developed in
primary culture were used, taking advantage that in this in vitro
system brown preadipocytes differentiate well under controlled conditions, thus allowing controlled studies of the expression and
function of key genes in response to regulatory signals. We centered
our study on the influence of the cis-9, trans-11
CLA, trans-10, cis-12 CLA, and cis-9,
cis-12 LA as the common polyunsaturated fatty acid chosen as
a reference. Under this influence, we investigated the expression of
the uncoupling proteins UCP1 and UCP2 and other key genes involved in
the regulation of cell differentiation and facultative thermogenesis
capacity in both the absence and presence of noradrenergic stimulation.
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MATERIALS AND METHODS |
Chemicals.
LA (>99% cis-9, cis-12 octadecadienoic acid)
and CLA (a mixture of cis and trans
octadecadienoic acids with the reported isomer content 41%
cis-9, trans-11 CLA and trans-9,
cis-11 CLA; 44% trans-10, cis-12 CLA;
and lesser proportions of other CLA isomers) were obtained from Sigma
(Madrid, Spain). cis-9, trans-11 CLA (>98%) was
obtained from Calbiochem (Darmstadt, Germany) and trans-10, cis-12 CLA (>98%) was obtained from Matreya (Pleasant Gap,
PA). Other cell culture reagents were supplied by Sigma, and routine chemicals were from Merck (Barcelona, Spain) and Panreac (Barcelona, Spain).
Cell culture, treatment, and harvesting.
Primary cultures of brown adipocytes were started with precursor cells
from cervical, axillary, and interscapular brown fat tissue of
4-wk-old, male NMRI mice (obtained from CRIFFA, Barcelona, Spain), as
described earlier (3). Pooled final cell suspension (0.2 ml) was inoculated per well (35-mm-diameter wells) and contained 1.8 ml
of culture medium consisting of DMEM supplemented with 10% newborn
calf serum, 4 nM insulin (Actrapid, Novo Industries, Denmark), 4 mM
glutamine, 10 mM HEPES, 25 µg/ml sodium ascorbate, and 50 IU
penicillin/50 mg streptomycin per milliliter. The cells were grown at
37°C and 8% CO2 in air. The medium was changed the day
after plating and on days 3 and 5. From day
6 onward, the cells fully differentiated into brown adipocytes.
Cells in culture were treated for a period of 24 h (from day
6 to day 7 of culture) with the different LA isomers,
freshly dissolved in ethanol, at doses indicated in Figs. 1-5,
alone or with a single dose of NE (1 µM; freshly dissolved in water).
Control cells received the same volume of ethanol or water only. All
experiments were performed at least two times.
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Fig. 1.
Cell morphology and lipid droplet shape in cultured brown
adipocytes. Phase-contrast micrographs of primary brown adipocytes
(A, original magnification ×100) and of oil red O-stained
primary brown adipocytes (B, original magnification ×320).
Cells were grown in culture and treated from day 6 to
day 7 of culture with 20 mg/l of different linoleic acid
isomers, alone or with a single dose of norepinephrine (NE). Control
cells received the same volume of ethanol or water only. On day
7 of culture, cells were oil red O stained and
photographed.
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Fig. 2.
Representative Northern blots for uncoupling protein
(UCP) 1, UCP2, and 3-adrenergic receptor (AR) mRNAs in
cultured brown adipocytes (A) and representative Western
blot for UCP1 (B). Cells were treated from day 6 to day 7 of culture with 20 mg/l of different linoleic acid
isomers, alone or with NE. Control cells received the same volume of
ethanol or water only. Total RNA (15 µg) was loaded per lane for
specific determination of the mRNA levels, using 18S rRNA as a control,
and 15 µg of whole cell lysate was used for immunoblot analysis.
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Fig. 3.
Cell proliferation in cultured brown adipocytes. Cells
were treated from day 2 to day 3 of culture with
20 mg/l of different linoleic acid isomers. Control cells received the
same volume of ethanol only. Twenty hours before detection assay, they
were labeled with 10 µM bromodeoxyuridine (BrdU), and its
incorporation into DNA was measured by an ELISA kit. Data are
means ± SE of 2 separate experiments. Significant differences
were tested by one-way ANOVA and least-significant difference (LSD)
post hoc comparisons (P < 0.05). FA, effect of fatty
acid treatment. Values not sharing a common letter were statistically
different.
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Fig. 4.
Dose-dependent effect on UCP1 mRNA expression in cultured
brown adipocytes. Cells were treated from day 6 to day
7 of culture with increasing doses of cis-9,
trans-11 CLA ( ) or trans-10, cis-12 CLA
( ) in the presence of noradrenergic stimulus (1 µM
NE). Control cells received the same volume of ethanol only. The
expression levels of the UCP1 mRNA were analyzed by Northern blotting
and normalized to the expression of 18S rRNA. Data represent means ± SE of at least 3 separate experiments and are expressed relative to
the mean value of the 10 µM NE-treated cells, which were set to 100. Significant differences were tested by ANOVA and LSD post hoc
comparison (P < 0.05). FA, effect of fatty acid
treatment. Values in the same line not sharing a common letter were
statistically different.
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Fig. 5.
Representative RT-PCR for leptin mRNA in cultured brown adipocytes.
Cells were treated from day 6 to day 7 of culture
with increasing doses of different linoleic acid isomers. Control cells
received the same volume of ethanol only. Total RNA (0.5 µg) was used
to semiquantify the mRNA levels, using the housekeeping gene -actin
as an internal control. Mk, 100-bp DNA leader (Invitrogen, Barcelona,
Spain).
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At the indicated time, the culture medium was removed, and the cells
were rinsed two times with ice-cold PBS (9.1 mM dibasic sodium
phosphate, 1.7 mM monobasic sodium phosphate, and 150 mM NaCl, pH 7.4)
and scraped in PBS. The cell suspension was pelleted, resuspended in a
minimal volume of PBS, and homogenized in a small well-fitting
glass-glass hand-operated homogenizer (10 strokes) before analysis.
For mRNA experiments, culture medium was removed, and the cells were
immediately scraped in Tripure reagent (Roche, Barcelona, Spain) to
isolate total RNA according to the instructions of the manufacturer.
The total RNA was then stored at 
70°C until analysis.
Bromodeoxyuridine labeling and detection assay.
Brown adipocytes were started and cultured as described above but in a
microtiter plate (96 wells). On day 2, preadipocytes were
treated with 20 mg/l of different LA isomers, freshly dissolved in
ethanol. Control cells received the same volume of ethanol. After
4 h, 10 µM bromodeoxyuridine was added. Finally, on day 3, cell proliferation was measured using the
5-bromo-2'-deoxyuridine labeling and detection ELISA kit (Roche)
following the manufacturer's instructions.
Oil red O staining.
Dishes used for oil red O staining were washed two times with PBS and
fixed by 10% formaldehyde in PBS for 15 min. Fixed cells were rinsed
with PBS and stained with oil red O-filtered solution (3 mg/ml in
isopropyl alcohol) for 1 h. Cells were then washed with water and
visualized with a Zeizz phase-contrast microscope (original
magnification ×320).
Protein and triacylglyceride content.
Protein concentration in cell lysates was measured by the method of
Bradford (5). Triacylglyceride concentration was
determined enzymatically using a commercial kit (procedure no. 336;
Sigma Diagnostics).
Cytochrome C oxidase activity determination.
Cytochrome c oxidase activity was measured from fresh cell
lysates by a spectophotometric method (47) to monitor
changes in absorbance during the oxidation of reduced ferricytochrome at 37°C.
Northern blot analysis.
Total RNA (15 µg) obtained from culture cells was denatured with
formamide/formaldehyde, resolved by agarose gel electrophoresis, and
then transferred to a Hybond nylon membrane and fixed with ultraviolet
light (20). 3-mRNA, UCP1-mRNA, UCP2-mRNA,
and 18S rRNA, as internal control, were analyzed sequentially on the
same membrane, in the above order, by a chemiluminiscence procedure based on the use of synthetic mouse-specific antisense oligonucleotide probes (37) end labeled with digoxigenin, essentially as
in the protocols provided by Roche. The technique was first described by Trayhurn et al. (43) in 1994. In short, fixed membranes
were prehybridized at 42°C for 15 min in DIG-Easy Hyb and then were hybridized with the corresponding oligonucleotide probe in DIG-Easy Hyb
at 42°C overnight. The membranes were blocked and incubated first
with an anti-digoxigenin-alkaline phosphatase conjugate and then with
the chemiluminiscent substrate CDP-Star. Finally, membranes were
exposed to Hyperfilm ECL (Amersham, Barcelona, Spain). Bands in films
were analyzed by scanner photodensitometry and quantified using the
BioImage program (Millipore, Bedford, MA). Stripping in between
analysis was performed by exposing the membranes to boiling 0.1% SDS.
Western blot analysis.
Proteins (15 µg) of whole cell lysates were fractionated by 12.5%
SDS-PAGE and electrotransferred to a nitrocellulose filter, as
previously described (3). Ponceau-S staining (0.1% in 5% acetic acid) was performed to check equal loading/transfer. Blocking and development of the immunoblots were perfomed using an enhanced chemiluminiscence Western-blotting analysis system (Amersham). Rabbit
polyclonal anti-UCP1 (Alpha Diagnostics, San Antonio, CA) was used as
primary antibody. Bands in film were analyzed by scanner photodensitometry and quantified using the BioImage program (Millipore).
RT-PCR analysis.
To semiquantify the levels of leptin, PPAR-2, ADD1, and
C/EBP- mRNAs we developed a RT-PCR assay, using the housekeeping gene -actin as an internal control, as previously described
(35). In brief, the 0.5 µg total RNA was denatured at
65°C for 10 min and reverse transcribed in the presence of 50 pmol
random primers, using murine leukemia virus (MuLV) reverse
transcriptase (Perkin-Elmer, Madrid, Spain) at 42°C for 15 min in a
Perkin-Elmer 2400 Thermal Cycler. After the reaction, the RT
medium (10%) was added to a PCR mix containing Taq DNA
polymerase (Promega, Lyon, France) and 2.5 pmol of -actin and 10 pmol of specific primers (for leptin, PPAR-2, or ADD1)
or 10 pmol of -actin and 10 pmol of specific primers (for C/EBP-;
see Ref. 35). The reaction mixture was first heated to
95°C for 2 min to denature the cDNA. This was followed by 30-36
cycles of denaturation at 95°C for 15 s, annealing at
59-60°C for 15 s, and extension at 72°C for 30 s,
with an additional extension at 72°C for 7 min after the last cycle.
The PCR products were separated in 3% agarose gel (MS-8; Pronadisa,
Madrid, Spain) in 0.5× Tris-borate-EDTA buffer, stained with ethidium
bromide, and visualized using an image-recording system (Gelprinter;
TDI, Madrid, Spain). The densities of the target bands were then
quantified using an image processing and analyzing program (BioImage; Millipore).
Statistical analysis.
Data were expressed as means ± SE. The effect of fatty acid
treatment, NE treatment, and its interaction (fatty acid × NE) on
the studied parameters was tested using two-way ANOVA; contrasts between means were assessed by least-significant difference or Student's t-test post hoc comparisons. The analyses were
performed with SPSS for windows (SPSS, Chicago, IL).
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RESULTS |
trans-10, cis-12 CLA reduced the NE-induced thermogenic capacity of
brown adipocytes, whereas both cis-9, trans-11 CLA and LA tended to
enhance it.
From day 6 onward, cultured brown fat cells were fully
differentiated as assessed by the presence of high levels of
cytoplasmic lipids (see Fig.
1A, control cells), by the
expression of key adipogenic transcription factors (see Table 4), and
by their capacity to induce UCP1 mRNA expression after noradrenergic
stimulus, from nondetectable levels to a maximum at 10 µM NE (see
Table 2 and a representative Northern and Western blot in Fig.
2).
Brown adipocytes (in both control and NE-stimulated conditions) treated
with the two CLA isomers and LA tended to have higher protein,
triacylglyceride, and cytochrome c oxidase activity than nontreated cells, probably as a consequence of the higher nutrient availability and utilization, principally for LA and
cis-9, trans-11 CLA (Fig. 1A
and Table 1). However, the notable
differences in lipid droplet shape observed between the two CLA-isomers
in non-NE-stimulated cells (as seen in Fig. 1B by oil red O
specific staining) would indicate some alteration in lipid
metabolism. In addition, we found that LA- and
cis-9, trans-11 CLA-, but not
trans-10, cis-12 CLA-, treated preadipocytes
showed higher rates of proliferation than nonfatty acid-treated cells,
tested by bromodeoxyuridine incorporation into DNA (Fig.
3).
No fatty acid studied induced the expression of UCP1 mRNA in cultured
brown adipocytes in the absence of NE in the cell culture media.
However, both LA and cis-9, trans-11 CLA
treatment tended to enhance the mRNA expression of UCP1 induced by 1 µM NE, whereas addition of trans-10, cis-12 CLA
or the CLA mixture markedly reduced the mRNA expression of UCP1 induced
by 1 µM NE (see Table 2 and a
representative Northern blot in Fig. 2A). We found a similar expression pattern of UCP1 protein levels in response to fatty acid
treatments (see a representative Western blot in Fig. 2B). Both cis-9, trans-11 CLA and
trans-10, cis-12 CLA treatments showed a
dose-dependent effect on UCP1 mRNA expression induced by 1 µM NE, as
seen in Fig. 4. In addition, UCP2 mRNA
behaved similarly to UCP1 fatty acid treatment: no effects on basal
UCP2 mRNA levels and reduced UCP2 mRNA expression induced by 1 µM NE
in the presence of trans-10, cis-12 CLA (see
Table 1 and a representative Northern blot in Fig. 2A).
We also investigated the expression of 3-AR, which has
been shown to mediate the stimulatory action of NE on UCP synthesis and
activity in mature adipocytes, to further study the brown adipocyte
thermogenic activity. Our results showed that treatment with either
cis-9, trans-11 CLA, trans-10,
cis-12 CLA, or its mixture, but not LA, downregulated mRNA levels
of 3-AR in both basal and NE-stimulated conditions
(Table 2 and Fig. 2, a representative Northern blotting). We observed
that both cis-9, trans-11 CLA and
trans-10, cis-12 CLA isomers produced a
dose-dependent response with a more pronounced response in the
treatment with the trans-10, cis-12 CLA isomer
(results not shown).
trans-10, cis-12 CLA reduced the expression of leptin in cultured
brown adipocytes.
Treatment with trans-10, cis-12 CLA
dramatically downregulated the basal mRNA expression of leptin in
differentiated brown adipocytes in culture, whereas both LA and
cis-9, trans-11 CLA tended to increase leptin
mRNA levels (see Table 3). As shown in
Fig. 5, trans-10,
cis-12 CLA reduced the leptin mRNA levels in a dose-dependent
manner. Moreover, the trans-10, cis-12 CLA effect
on leptin mRNA was also observed under NE stimulus (Table 3).
trans-10, cis-12 CLA reduced the basal expression of the key
adipocyte transcription factors PPAR-2 and ADD1 in
mature brown adipocytes and inhibited the upregulation of
PPAR-2 and C/EBP- in NE-stimulated cells.
PPAR-2, C/EBP-, and ADD1 are the key adipocyte
transcription factors that control thermogenesis and lipid
metabolism in brown adipocytes and are well expressed in cultured brown
adipocytes. trans-10, cis-12 CLA reduced the
basal mRNA expression of ADD1 and PPAR-2, whereas
addition of cis-9, trans-11 CLA increased the
mRNA expression of C/EBP- (Table 4).
LA also tended to increase C/EBP- mRNA (see Table 4).
Under stimulation by NE, brown fat cells showed an upregulation of the
PPAR-2 and C/EBP- mRNA levels (see Table 4). However, addition of trans-10, cis-12 CLA blocked the
induction of PPAR-2 and C/EBP- mRNA expression by NE.
On the other hand, addition of cis-9, trans-11
CLA or LA did not disturb the induction of these two transcription
factors, yet both of them tended to enhance the expression of the
transcription factor studied (see Table 4).
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DISCUSSION |
Our results showed opposite effects for the two main CLA isomers
on thermogenic capacity, as well as on proliferation, morphology, and
the expression of key adipogenic specific genes controlling thermogenesis and lipid metabolism in cultured brown adipocytes. This
study has examined for the first time the specific separate effects of
cis-9, trans-11 CLA, trans-10, cis-12
CLA, and LA treatment on thermogenic capacity, in terms of UCP1, UCP2,
and 3-AR expression, in primary cultured brown
adipocytes, stimulated or not with NE, the main physiological regulator
of these cells.
trans-10, cis-12 CLA treatment reduces adipogenesis coupled to a
defective thermogenesis.
We showed that treatment of brown adipocytes with trans-10,
cis-12 CLA, at doses found in sera from rodents fed
CLA-supplemented diets, i.e., ~20 mg/l (31), reduced the
expression of UCP1 mRNA induced by NE and, to a lesser extent, of UCP2
(Table 2). These changes were consistent with a blockage of induction
of C/EBP- and PPAR-2 mRNA expression. These genes are
known to be involved in the expression of UCP1 (39). Thus
thermogenic capacity became depressed in brown adipocytes by
trans-10, cis-12 CLA treatment at the level of gene
expression. A downregulation of mRNA levels of 3-AR was
also observed, but it also occurred for cis-9, trans-11 CLA
treatment, indicating that differences in the action of both isomers
were not related to this receptor (Tables 2 and 4).
Lipid metabolism in brown adipocytes was also altered by
trans-10, cis-12 CLA; it reduced lipid droplet accumulation
and leptin mRNA expression when compared with cis-9,
trans-11 CLA or LA treatments, which tended to increase both lipid
content and leptin mRNA expression (Figs. 1 and 5). It has recently
been shown that consumption of a mixture of CLA reduces plasma leptin
concentration, but not when using a cis-9, trans-11
CLA-enriched diet (40). Moreover, feeding mice a diet
supplemented with trans-10, cis-12 CLA reduces body fat
gain, serum leptin, and adipocyte leptin mRNA expression without
affecting food intake(21), presumably reflecting the
treatment-induced reduction in adiposity and/or volume of adipocytes
(48). In addition, Kang and Pariza (21) also
showed that trans-10, cis-12 CLA reduces leptin expression
and secretion in cultured 3T3-L1 adipocytes. In our results, changes in
lipid droplet morphology were shown to be associated with lower basal
expression levels of the PPAR-2 and ADD1 mRNAs, which
control the expression of genes involved in adipogenesis and lipid
metabolism (39). These effects seem to be characteristic
of adipose lineage, since CLA-supplemented diet-fed animals show a
downregulation of PPAR-2 and ADD1 mRNA in parametrial
white adipose tissue (44). Moreover, a downregulation of
PPAR-2 and C/EBP- in cultured 3T3-L1 adipocytes has
been observed after CLA treatment (6).
In addition, it could be deduced from our results on UCP expression
that the effect of trans-10, cis-12 CLA
predominated over the effects of cis-9, trans-11 CLA when
both isomers are equally present in a mixture. The reduction of UCP1
and UCP2 mRNA expression induced by the CLA mixture containing both
isomers is directly related to its trans-10,
cis-12 CLA content (Table 2).
cis-9, trans-11 CLA treatment enhances UCP1 thermogenic capacity.
On the other hand, treatment with either cis-9, trans-11 CLA
or LA showed very different effects than those of
trans-10, cis-12 CLA. cis-9, trans-11
CLA and LA enhanced the mRNA expression of UCP1 induced by NE (Table
2), an effect reported earlier for fatty acid diet supplementation
(38, 41). Both are ligands of PPARs (22) and
could transactivate the expression of UCP1 mRNA. CLA is reported to
interact with the three PPAR isoforms (PPAR-, PPAR-, and
PPAR-; see Refs. 17 and 28). Nevertheless, there is
evidence that the influence of CLA on body composition may function
independently of PPAR- (33) and that dietary CLA has
profound glucose-, insulin-, and free fatty acid-lowering properties
that may be mediated by PPAR- (17). In addition, cis-9, trans-11 CLA and, to a lesser extent, LA (probably
through the trans-activation of PPAR-2)
induced the expression of C/EBP-, which is known to be important for
leptin expression and other terminal adipocyte differentiation
processes (18). Thus cis-9, trans-11 CLA and LA
could enhance brown adipocyte growth by inducing preadipocyte
proliferation (see Fig. 3) and lipid accumulation (Fig. 1). These
effects on recruitment were consistent with an increase in nutrient
availability and activation of the differentiation cascade through
PPARs and ADD1. Under noradrenergic stimulus, both cis-9,
trans-11 CLA and LA could enhance thermogenesis by increasing fuel
availability to the respiratory chain, directly inducing UCP1 activity,
and enhancing expression of all UCPs (29). Hence, the
increased total energy expenditure in CLA-fed animals (44-46) could be explained, at least in part, by
cis-9, trans-11 CLA content. Other mechanisms proposed to
explain an enhancement of thermogenesis, such as the induction of UCP2
mRNA levels in white adipose tissues (40, 44) and skeletal
muscle (40), appear more unlikely, since the physiological
role of this new UCP in adaptative thermogenesis has not been confirmed
when the corresponding gene knockout mice have been obtained
(1). Other studies have described an increase in total
energy expenditure by feeding CLA without an increase in UCP gene
expression in adipose tissues and muscle (45). In all
these studies, the relevance of the dose, time, and CLA preparation
used in CLA treatment have been pointed out, but no specific mechanism
for enhanced thermogenesis has been shown. Moreover, Park et al.
(32) described a tendency to reduce body weight in animals
fed several CLA-enriched diets for 4 wk. Animals fed with enriched
trans-10, cis-12 CLA diet and CLA mixture exhibit a higher
reduction of body weight, probably associated with the observed
reduction in food intake. However, in the case of animals fed a
cis-9, trans-11 CLA-enriched diet, there is a
reduction in body weight with no changes in food intake, in which
facultative thermogenesis could take place.
In conclusion, our results prove opposite effects of CLA isomers on
thermogenic capacity and on both cell proliferation and differentiation
in cultured brown adipocytes; trans-10, cis-12 CLA treatment
reduces adipogenesis coupled to a defective thermogenesis, and
cis-9, trans-11 CLA treatment enhances UCP1
thermogenic capacity. Both effects could explain, at least in part, the
changes in body fat content and energy metabolism induced in animal
models by feeding a CLA-supplemented diet. Furthermore, the effects of
the trans-10, cis-12 CLA isomer on adipogenesis
and thermogenesis superimposes the cis-9,
trans-11 CLA effect. Our findings could have important nutritional
implications, since they indicate that, despite high total dietary fat
consumption, specific dietary fatty acid consumption, including
specific compounds, i.e., the family of CLAs, may have profound effects
on adiposity and energy expenditure. Hence, this should be taken into
account in future managements of obesity and insulin resistance.
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ACKNOWLEDGEMENTS |
This work was supported by the Spanish Government (Dirección
General de Investigación, BFI2000-0988-C06, and Programa de Promoción a la Investigación Biomedica y en Ciencias de la
Salud, Ministerio de Sanidad y Consumo, FIS01/1379) and the European Union (Cooperation in Space and Technology action 918 and Grant QLRT-2001-00183). E. Rodríguez was the recipient of a
doctoral fellowship from the Spanish Government (Ministerio de
Educación, Cultura y Deportes).
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FOOTNOTES |
Address for reprint requests and other correspondence: A. Palou, Dept. de Biologia Fonamental i Ciències de la Salut,
Universitat de les Illes Balears, Cra Valldemossa, km 7.5, 07071 Palma
de Mallorca, Spain (E-mail:
andreu.palou{at}uib.es).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 7, 2002;10.1152/ajpregu.00637.2001
Received 26 October 2001; accepted in final form 6 February 2002.
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