Antiobesity mechanisms of action of conjugated linoleic acid.
ABSTRACT Conjugated linoleic acid (CLA), a family of fatty acids found in beef, dairy foods and dietary supplements, reduces adiposity in several animal models of obesity and some human studies. However, the isomer-specific antiobesity mechanisms of action of CLA are unclear, and its use in humans is controversial. This review will summarize in vivo and in vitro findings from the literature regarding potential mechanisms by which CLA reduces adiposity, including its impact on (a) energy metabolism, (b) adipogenesis, (c) inflammation, (d) lipid metabolism and (e) apoptosis.
- SourceAvailable from: Begoña Olmedilla-Alonso (OR Olmedilla)[Show abstract] [Hide abstract]
ABSTRACT: RESUMEN and ABSTRACT Por múltiples razones los productos cárnicos son alimentos excepcionalmente adecuados para introducir en la dieta diversos compuestos bioactivos sin modificar los hábitos de consumo. En los últimos años se ha producido un notable desarrollo de productos cárnicos diseñados como potencialmente funcionales. En el marco de la alimentación funcional, este artículo proporciona una panorámica general acerca de las razones que impulsan y justifican su obtención haciendo especial énfasis en: a) los aspectos a considerar en su diseño con el propósito de poder realizar declaraciones nutricionales y alegaciones de propiedades saludables; b) las estrategias empleadas para optimizar la presencia de ingredientes funcionales, favoreciendo la presencia de compuestos bioactivos beneficiosos y limitando la de aquellos otros con implicaciones negativas para la salud, y c) los procedimientos para demostrar una relación entre la ingesta de productos cárnicos potencialmente funcionales con un efecto beneficioso sobre la salud y de qué manera se reflejan tales estudios en la bibliografía. Abstract For a number of reasons, meat products are an exceptionally adequate means for introducing different bioactive compounds into the diet without modifying eating habits. In recent years, there has been a notable development of meat products designed as potentially functional foods. Within the framework of the functional food, this article provides a general view of the reasons that motivate and justify their formulation, with special emphasis on: a) aspects to be considered in their design in order to be able to make nutrition claims and statements concerning their health-promoting properties; b) the strategies employed to optimize the presence of functional ingredients, favoring the presence of beneficial bioactive compounds and limiting others with negative consequences for our health, and c) the procedures for demonstrating a relationship between the consumption of potentially functional meat products with beneficial effects on health and the way in which these studies are reflected in the literature.Nutricion hospitalaria: organo oficial de la Sociedad Espanola de Nutricion Parenteral y Enteral 06/2014; · 1.31 Impact Factor
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ABSTRACT: This study aims to evaluate the effects of dietary conjugated linoleic acid (CLA) and phytosterol supplementation and their combination added to high fat diet to verify their action on the satiety hormones, food intake, weight loss and peripheral glucose intolerance and insulin resistance in healthy rats. Twenty-one-day-old male healthy Sprague–Dawley rats were divided into five groups (n=5): Standard group (P), Standard High-Fat group (HF), and three high-fat groups fed diets with 2% CLA added (HC), 2% phytosterol (HP) and 2% CLA plus 2% phytosterol (HS). The standard diet was AIN-93G with 12% protein and 4% vegetable oil; the high-fat diet contained 12% protein and 35% fat (4% vegetable oil and 31% animal fat). The experimental period lasted 8 weeks. The groups receiving supplements showed a significant reduction of retroperitoneal and epididymal adipose tissue as compared to the HF group (p<0.05). However, there was no improvement in insulin resistance and glucose in either supplemented group (p<0.05). The leptin decreased only in the groups that were receiving CLA (p<0.05). All supplemented groups showed a reduction in diet consumption, but the groups that received the supplements showed weight reduction compared to the HF group (p<0.05). The animals that received the supplements alone or in combination showed reduced fat mass and the effect was potentiated in the group HS, but there was no improvement in ghrelin, adiponectin insulin and glucose resistance. The supplements alone or in combination promoted reduction of food intake and also promoted weight loss in experimental animals.
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ABSTRACT: Lipid-soluble molecules share several aspects of their physiology due to their common adaptations to a hydrophilic environment, and may interact to regulate their action in a tissue-specific manner. Dietary conjugated linoleic acid (CLA) is a fatty acid with a conjugated diene structure that is found in low concentrations in ruminant products and available as a nutritional supplement. CLA has been shown to increase tissue levels of retinol (vitamin A alcohol) and its sole specific circulating carrier protein retinol-binding protein (RBP or RBP4). However, the precise mechanism of this action has not been elucidated yet. Here, we provide a summary of the current knowledge in this specific area of research and speculate that retinol and CLA may compete for catabolic pathways modulated by the activity of PPAR-α and RXR heterodimer. We also present preliminary data that may position PPAR-α at the crossroads between the metabolism of lipids and vitamin A.Nutrients 01/2014; 6(3):1262-72. · 2.07 Impact Factor
REVIEWS: CURRENT TOPICS
Antiobesity mechanisms of action of conjugated linoleic acid
Arion Kennedya, Kristina Martineza, Soren Schmidtb, Susanne Mandrupb, Kathleen LaPointa,
aDepartment of Nutrition, University of North Carolina Greensboro, PO Box 26170, Greensboro, NC 27402-6170, USA
bDepartment of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
Received 5 May 2009; received in revised form 6 August 2009; accepted 19 August 2009
Conjugated linoleic acid (CLA), a family of fatty acids found in beef, dairy foods and dietary supplements, reduces adiposity in several animal models of
obesity and some human studies. However, the isomer-specific antiobesity mechanisms of action of CLA are unclear, and its use in humans is controversial. This
review will summarize in vivo and in vitro findings from the literature regarding potential mechanisms by which CLA reduces adiposity, including its impact on
(a) energy metabolism, (b) adipogenesis, (c) inflammation, (d) lipid metabolism and (e) apoptosis.
© 2010 Elsevier Inc. All rights reserved.
Keywords: CLA; Obesity; Adipose tissue; Energy metabolism; PPARγ; Adipogenesis; Inflammation
Conjugated linoleic acid (CLA) refers to a group of conjugated
octadecadienoic acid isomers derived from linoleic acid, a fatty acid
that contains 18 carbons and 2 double bonds in cis configuration at
the 9th and 12th carbons (i.e., cis-9,cis-12 octadecadienoic acid).
Microbes in the gastrointestinal tract of ruminant animals convert
linoleic acid into different isoforms of CLA through biohydrogenation.
This process changes the position and configuration of the double
bonds, resulting in a single bond between one or both of the two
double bonds [i.e., cis-9,trans-11 (9,11) or trans-10,cis-12 (10,12)
Commercial preparations of CLA are made from the linoleic acid of
safflower or sunflower oils under alkaline conditions. This type of
processing yields a CLA mixture containing approximately 40% of the
9,11 isomer and44% ofthe 10,12 isomer (reviewedin Parizaet al. ).
Commercial preparations also contain approximately 4–10% trans-9,
trans-11 CLA and trans-10,trans-12 CLA, as well as trace amounts of
The 9,11 isomer, also known as rumenic acid, is the predom-
inant form of CLA found in naturally occurring foods. 9,11-CLA
comprises approximately 90% of CLA found in ruminant meats and
dairy products, and the 10,12 isomer comprises the remaining 10%.
Although several other isoforms of CLA have been identified (i.e.,
trans-9,trans-11, cis-9,cis-11, trans-10,trans-11 and cis-10,cis-12),
the 9,11 and 10,12 isomers appear to be the most biologically
The proportion of CLA ranges from 0.34% to 1.07% of the total fat in
dairy products, and from 0.12% to 0.68% of the total fat in raw or
processed beef products (reviewed in Dhiman et al. , Silveira et al.
 and Mendis et al. ). However, the CLA content of food is
dependent on several factors, including the season and the animal's
breed, nutritional status and age (reviewed in Dhiman et al. ). The
average daily intake of CLA is approximately 152–212 mg for
nonvegetarian women and men, respectively , and human serum
levels range from 10 to 70 μmol/L [7,8].
1.1. Antiobesity properties of CLA
CLA was initially discovered in 1987 by Ha et al. , and it was first
identified as an anticarcinogen. Subsequently, CLA was shown to
exhibit antiatherosclerotic (reviewed in Mitchell and McLeod )
and antiobesity (reviewed in Whigham et al. ) properties. Due to
the substantial rise in the prevalence of obesity over the past 30 years
, interest in CLA as a weight loss treatment has increased.
Supplementation with a CLA mixture (i.e., equal concentrations of the
10,12 and 9,11 isomers) or the 10,12 isomer alone decreases body fat
mass (BFM) in many animal studies and some human studies
(reviewed in Whigham et al.  and Wang and Jones ). Of the
two major isomers of CLA, the 10,12 isomer is specifically responsible
for the antiobesity effects [14–18].
1.2. CLA regulation of body weight
Park et al.  were the first to demonstrate that CLA modulated
body composition. In their study, male and female mice given a 0.5%
(wt/wt) CLA mixture had 57% and 60% lower BFM, respectively, than
controls. Other researchers have subsequently demonstrated that
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Journal of Nutritional Biochemistry 21 (2010) 171–179
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CLA supplementation consistently reduces BFM in mice, rats and
pigs [20–24]. For example, dietary supplementation with 1% (wt/
wt) CLA mixture for 28 days decreased body weight and
periuteral white adipose tissue (WAT) mass in C57BL/6J mice
. Similarly, a 1.0–1.5% (wt/wt) mixture of CLA for 3–4 weeks
decreased body weight and WAT mass in male ob/ob  and ICR
 obese mice.
Studies investigating CLA's effects on BFM reduction in humans
have produced less consistent results. Whereas some studies show
that CLA decreases BFM and increases lean body mass (LBM)
(reviewed in Wang and Jones  and House et al. ), others
have shown no effect of CLA supplementation on body composition in
humans (reviewed in Wang and Jones ). For example, supple-
mentation of a CLA mixture in overweight and obese people (3–4 g/
day for 24 weeks) decreased BFM and increased LBM . On the
other hand, supplementation of CLA mixture in yogurt in healthy
adults (3.76 g/day for 14 weeks) had no effect on body composition
. In addition, Larsen et al.  investigated the potential role of
CLA in preventing body weight regain in moderately obese subjects
who had lost approximately 10 kg after an 8-week dietary
intervention on a low-calorie diet. Supplementation for 1 year with
a CLA mixture did not prevent body weight regain compared to
controls. Finally, supplementation with 3.2 g/day of a CLA mixture
decreased total BFM and trunk fat compared to placebo in overweight
subjects, but not in obese subjects . These contradictory findings
among human studies may be due to the following differences in
experimental design: (a) CLA isomer combination versus individual
isomers, (b) CLA dose and duration of treatment, and (c) the gender,
weight, age and metabolic status of the subjects.
The primary discrepancy between animal and human studies
appears to be the dose of CLA administered. For example, moderately
overweight humans with an average weight of 72.5 kg supplemented
with a CLA mixture (3.76 g/day for 14 weeks) experienced no
decrease in body weight, BMI or BFM . In contrast, C57BL/6 mice
supplemented with 1.5% (wt/wt) CLA mixture for 4 weeks weighed
significantly less and had reduced adiposity compared to controls
. However, while subjects in the human study received approx-
imately 0.05 g/kg body weight CLA, the mice received 1.07 g/kg body
weight CLA, which was 20 times the human dose based on body
weight. Supplementing humans with higher doses of CLA would
address this dosing issue.
Because CLA has the potential to reduce BFM when given at high
enoughdosesandis beingtakenasa supplement forthatpurpose,itis
important to understand its mechanism of action. Therefore, this
review will examine potential mechanisms by which the CLA mixture
or 10,12-CLA alone reduces adiposity, with particular emphasis on its
effects on WAT. Potential mechanisms to be discussed include
regulation of (a) energy metabolism, (b) adipogenesis, (c) inflam-
mation, (d) lipid metabolism and (e) apoptosis.
2. Antiobesity mechanisms of CLA
2.1. CLA regulation of energy metabolism
2.1.1. CLA decreases energy intake
Energy balance is a function of energy intake relative to energy
expenditure. When energy intake exceeds energy expenditure, body
weight and BFM increase and vice versa. Accordingly, potential
mechanisms by which CLA reduces BFM include decreasing energy
intake or increasing energy expenditure. Park et al.  demon-
strated that mice supplemented with a CLA mixture or enriched
10,12-CLA for 4 weeks reduced their food intake. A number of
subsequent studies in rodents produced similar results [16,33–36].
No study to date has demonstrated that CLA decreases food intake
in humans [37–40].
A mechanism for this reduced food intake was suggested by So et
al. , who reported that food intake was reduced by 23.6% in mice
fed a low-fat diet supplemented with 10,12-CLA. In this study, mice
receiving 10,12-CLA had a decreased gene expression ratio of
proopiomelanocortin to neuropeptide Y in the hypothalamus. These
results suggest that CLA exerts an effect on hypothalamic appetite-
regulating genes. In support of this hypothesis, injection of mixed
isomers of CLA to the rat hypothalamus reduced the expression of
neuropeptide Y and agouti-related protein — neuropeptides that
robustly increase food intake . Alternatively, CLA supplementa-
tion may reduce food intake by affecting the palatability of the diet;
however, so far, there have been no reports supporting this
A number of studies have reported reduced adiposity without
changes in energy intake following administration of a CLA mixture in
mice [42–45]. For example, supplementation of mice with a CLA
mixture for 42 days decreased total body weight without reducing
food intake . These data indicate that CLA effects on body fat are
not solely dependent on reduction in food or reduction in energy
intake. Thus, althoughseveralstudies showthat CLA decreases energy
intake, others show no effect, suggesting that CLA can decrease body
fat independent of reduction of energy intake.
2.1.2. CLA increases energy expenditure
Energy expenditure is a function of basal metabolic rate (BMR),
adaptive thermogenesis, and physical activity. CLA has been proposed
to reduce adiposity by elevating energy expenditure via increased
BMR, thermogenesis or lipid oxidation in animals [33,43–47]. For
example, in BALB/c male mice fed mixed isomers of CLA for 6 weeks,
body fat was decreased by 50% compared to controls and was
accompanied by increased BMR . Enhanced thermogenesis may
be associated with an up-regulation of uncoupling proteins (UCPs),
which facilitate proton transport over the inner mitochondrial
membrane, thereby diverting energy from ATP synthesis to heat
production. UCP1 was the first member of the family to be isolated
and is exclusively expressed in brown adipose tissue. UCP3 is
expressed in muscle and in a number of other tissues. UCP2 is
expressed in a variety of tissues, including WAT, and is the most
highly expressed UCP. Supplementation with a CLA mixture or 10,12-
CLA in rodents has been shown to induce UCP2 transcription in WAT
[27,35,48–50], but whether this plays a role in energy dissipation is
unclear. CLA also increased the expression of another mitochondrial
protein, carnitine palmitoyltransferase 1 (CPT1), in WAT of 10,12-
CLA-treated mice [50,51]. CPT1 is involved in mitochondrial fatty acid
uptake and catalyzes the rate-limiting step of fatty acid oxidation.
Consistent with these findings, 10,12-CLA increased β-oxidation in
differentiating 3T3-L1 mouse preadipocytes . Similarly, CLA
supplementation has been shown to increase UCP expression and
β-oxidation in rodent muscle and liver [35,53–57].
On the other hand, results from human studies concerning CLA
regulation of energy expenditure are mixed. For example, a recent
investigation of human supplementation with a CLA mixture (3.9 g/
day for 12 weeks) revealed no change in BMR or BFM . Similar
results have been reported in other studies of humans supplemented
with a CLA mixture [37,58]. In contrast, healthy moderately
overweight humans consuming a CLA mixture in yogurt (3.76 g/day
for 14 weeks) exhibited higher BMR, although body weight was not
affected . Similarly, supplementation with a CLA mixture for 13
weeks increased the resting metabolic rate and fat-free mass in
human subjects without a corresponding effect on BFM . Thus far,
only one human study has demonstrated both increased energy
expenditure and decreased body weight in humans. In this study by
Close et al. , subjects supplemented with a CLA mixture (4 g/day
for 6 months) had decreased body weight and exhibited increased fat
oxidation and energy expenditure while sleeping.
A. Kennedy et al. / Journal of Nutritional Biochemistry 21 (2010) 171–179
Other studies have demonstrated that CLA supplementation
increases LBM, which is associated with higher levels of energy
expenditure. For example, mixed CLA isomers (6.4 g/day for 12
weeks) increased LBM by 0.64 kg in healthy obese humans compared
to controls . Similarly, mice fed a 0.4% (wt/wt) CLA mixture
exhibited increased LBM compared to controls . Proposed
mechanisms by which CLA increases LBM occur via increased bone
or muscle mass, which is supported by evidence from rodent studies.
A 10-week 10,12-CLA supplementation [0.5% (wt/wt) mixed isomers]
increased bone mineral density and muscle mass in C57BL/6 female
mice . CLA supplementation is thought to increase bone mineral
density by up-regulating osteogenic gene expression and by down-
regulating osteoclast bone-resorbing activity [62,63]. Similarly, CLA
supplementation alone or with exercise increased bone mineral
density in middle-aged female mice compared to controls .
Alternatively, CLA may suppress the adipogenesis of pluripotent
mesenchymal stem cells (MSCs) in bone marrow andinstead enhance
their commitment to become osteoblasts (bone-forming cells).
Indeed, 10,12-CLA has been shown to preferentially promote the
differentiation of human MSCs into osteoblasts in culture . In
contrast, 9,11-CLA increased adipocyte differentiation and decreased
osteoblast differentiation. Consistent with these in vitro data, CLA
mixture supplementation of rats treated with corticosteroids, which
decrease muscle and bone mass, prevented reductions in LBM, bone
mineral density, and bone mineral content . Collectively, these
findings suggest that CLA may reduce adiposity through increased
energy expenditure via increased mitochondrial uncoupling and fatty
acid oxidation in WAT, or via increased muscle or bone mass.
However,the extentto which CLA regulatesBMRor LBM andhowthis
contributes to the reduction in body weight or fat in humans remain
to be determined.
2.2. CLA regulation of adipogenesis
2.2.1. CLA inhibits adipogenesis
The conversion of preadipocytes into adipocytes involves the
activation of key transcription factors such as peroxisome prolif-
erator-activated receptor γ (PPARγ) and CAAT/enhancer binding
protein (C/EBP). During the differentiation process, increased C/EBPβ
and C/EBPδ activity induces the transcription of C/EBPα and PPARγ,
the master regulators of adipocyte differentiation (Fig. 1). There is
much evidence showing that CLA suppresses preadipocyte differen-
tiation in animal [18,52,67–71] and human [72,73] preadipocytes.
10,12-CLA treatment has been reported to reduce adipogenesis and
lipogenesis specifically by attenuating PPARγ, C/EBPα, sterol regula-
tory element binding protein (SREBP) 1c, liver X receptor (LXR) α,
and adipocyte-specific fatty acid binding protein (aP2) expression
In rodents, 10,12-CLA supplementation decreased the expression
of PPARγ and its target genes [24,50,71,76]. In mature in vitro
differentiated primary human adipocytes or in mature 3T3-L1
adipocytes, 10,12-CLA treatment leads to a substantial decrease in
the expression and activity of PPARγ [18,77] and a decrease in PPARγ
target genes and lipid content . These and numerous other studies
show that 10,12-CLA specifically is not only able to inhibit but also
able to reverse the adipogenic process, and that this may in part be
mediated by suppression of PPARγ activity (Fig. 2). The decrease in
PPARγ target gene expression may be due to reduced PPARγ
expression or posttranslational inhibition of PPARγ activity per se.
Because PPARγ directly or indirectly induces its own expression,
decreased PPARγ activity would be expected to suppress PPARγ
expression, making it difficult to determine the level at which
inhibition occurs. Time-course experiments conducted in our labora-
tories, however, indicate that inhibition of PPARγ activity occurs prior
to a decrease in PPARγ expression , suggesting that inhibition of
PPARγ activity may be a primary event.
There are many mechanisms by which CLA may posttranslation-
ally regulate PPARγ. Transient transfection assays with ectopically
expressed PPARγ have been employed to assess CLA isomers as
potential ligands for PPARγ. In such assays, both CLA isomers have
been shown to only modestly activate PPARγ, even at high
concentrations. However, they are able to effectively inhibit the
action of full agonists such as rosiglitazone and darglitazone
[18,70,77,78], indicating that CLA may act as a low-affinity partial
agonist. Nonetheless, this mechanism cannot fully account for the
10,12-CLA-specific repression of PPARγ activity.
PPARγ activity may also be regulated by phosphorylation (Fig. 2),
which can be mediated by the mitogen-activated protein kinase
(MAPK) pathway [79–81]. Ser112 phosphorylation of PPARγ2, the
form of PPARγ requiredfor adipocyte differentiation, may decrease its
activity via ubiquitination and proteasome degradation , and via
reduction in both its ligand-dependent and its ligand-independent
transactivating functions [80,83–85]. We have demonstrated that 24-
h treatment with 10,12-CLA increases PPARγ phosphorylation 
without significantly decreasing its protein levels, suggesting that the
down-regulation of PPARγ target genes is due to decreased
transactivating function. Intriguingly, robust extracellular-signal-
regulated kinase (ERK) phosphorylation is also observed 24 h after
CLA stimulation, suggesting a role for ERK in PPARγ phosphorylation
and inactivation. Consistent with these data, we demonstrated that
ERK activation is a key player in CLA's suppression of adipogenic gene
expression and insulin-stimulated glucose uptake . Therefore, it is
tempting to speculate that CLA antagonizes PPARγ activity via
activation of MAPKs such as ERK, thereby leading to repression of
PPARγ target genes (Fig. 2).
Finally, CLA may interfere with PPARγ activity by virtue of its
proinflammatory effects on adipocytes (Fig. 3). We have shown that
10,12-CLA induces nuclear factor κB (NFκB) activation in adipocytes,
and that this induction leads to an increased expression of
proinflammatory cytokines [86,87]. In addition, NFκB or other
proinflammatory transcription factors may interfere directly with
PPARγ activation of target genes (Figs. 1–3). This will be discussed in
more detail below.
2.3. CLA increases inflammation
the ability to produce a number of proinflammatory cytokines. These
adipokines (i.e., cytokines produced by adipose tissue) can cause
insulin resistance (Fig. 4), thereby suppressing lipid synthesis and
increasing lipolysis in adipocytes (Fig. 5). Induction of these
inflammatory genes is dependent on various cellular kinases,
including MAPK, and is driven by transcription factors such as NFκB,
which have been reported to directly antagonize PPARγ (Figs. 1–3).
Tumor necrosis factor α (TNFα), in particular, exerts potent
Fig. 1. 10,12-CLA antagonizes the expression and activity of PPARγ and C/EBPα, master
regulators of adipocyte differentiation and maintenance. We propose that 10,12-CLA
impairs preadipocyte differentiation and maintenance of mature adipocytes by (a)
decreasing the expression of PPARγ and C/EBPα, and (b) activating inflammatory
proteins such as NFκB and MAPKs that antagonize PPARγ activity, thereby reducing the
expression of PPARγ target genes. TF = transcription factor.
A. Kennedy et al. / Journal of Nutritional Biochemistry 21 (2010) 171–179
antiadipogenic effects [88,89], and interleukin (IL) 1β and interferon
γ have been observed to induce delipidation of human adipocytes
. Treatment with 10,12-CLA has also been shown to increase the
expression or secretion of IL-6 and IL-8 from murine [24,50] and
human [73,77,86] adipocyte cultures, as well as TNFα and IL-1β,
thereby suppressing PPARγ activity and insulin sensitivity
In human subjects, 10,12-CLA supplementation also increases the
levels of inflammatory prostaglandins (PGs) [58,92]. For example,
women supplemented with mixed CLA isomers (5.5 g/day for 16
weeks) exhibited higher levels of C-reactive protein in serum and 8-
iso-PGF2αin urine. Accordingly, the expressionof cyclooxygenase
2, an enzyme involved in the synthesis of PGs, was elevated in
cultures of newly differentiated human adipocytes treated with
10,12-CLA . Furthermore, 10,12-CLA increased PGF2αsecretion
from human adipocytes .
Inflammatory PGs such as PGF2αhave been reported to inhibit
adipogenesis via phosphorylation of PPARγ by MAPKs  and via
induction of the normoxic activation of hypoxia-inducible factor-1
(HIF-1). HIF-1 decreases PPARγ and C/EBPα expression by up-
regulating the transcriptional repressor DEC1 [94,95]. In addition,
PGF2α may inhibit adipogenesis by activating proinflammatory
transcription factors that antagonize PPARγ.
Notably, data from our laboratory show that activation of ERK
and NFκB plays a critical role in 10,12-CLA's suppression of
adipogenic genes and insulin-stimulated glucose uptake [73,86].
The molecular mechanisms by which NFκB and other inflammatory
transcription factors inhibit PPARγ activity are not completely
understood, but results from a study on the bone marrow stromal
cell line ST2 suggest that NFκB interacts directly with PPARγ
preventing it from binding DNA [96,97]. In a different study using
chromatin immunoprecipitation, the DNA binding activity of PPARγ
did not appear to be affected by TNFα stimulation in 3T3-L1
adipocytes or human embryonic kidney 293 cells. Instead, suppres-
sion of PPARγ activity involved IKK activation, leading to IκBα
degradation and nuclear localization of histone deacetylase 3, a
component of the PPARγ corepressor complex [98,99]. NFκB may
also repress PPARγ activity via interaction with the DNA-bound
retinoid X receptor–PPARγ heterodimer, thereby interfering with
Taken together, these data suggest that 10,12-CLA antagonizes
PPARγ activity via inflammatory mediators such as MAPKs and NFκB
or via induction of inflammatory PG and adipocytokine production,
which in turn antagonize PPARγ activity.
2.4. CLA regulation of lipid metabolism
2.4.1. CLA suppresses lipogenesis
Storage of fatty acids such as triglycerides (TGs) is a major
function of adipocytes. Numerous proteins involved in lipogenesis,
such as lipoprotein lipase (LPL), acetyl-CoA carboxylase (ACC), fatty
acid synthase (FAS), and stearoyl-CoA desaturase (SCD), are
decreased by supplementation with mixed isomers of CLA or
Fig. 2. 10,12-CLA may antagonize PPARγ activity by (1) decreasing PPARγ gene
expression; (2) enhancing PPARγ degradation via phosphorylation, ubiquitination and
proteosome degradation; or (3) increasing NFκB activation, which impairs PPARγ DNA
binding and subsequent induction of adipogenic and lipogenic gene expression.
Fig. 3. 10,12-CLA activation of inflammatory proteins and induction of inflammatory
genes interfere with PPARγ transcriptional activation of target genes such as LPL,
adiponectin (AMP1), GLUT4 and aP2.
Fig. 4. 10,12-CLA-mediated insulin resistance is linked to (a) antagonism of PPARγ-
induced GLUT4 and adiponectin (AMP1) expression, and (b) induction of inflamma-
tory proteins and genes that decrease IRS-1-P (tyr) abundance, thereby reducing
GLUT4 translocation to the plasma membrane.
Fig. 5. 10,12-CLA increases lipolysis acutely and decreases lipogenesis chronically by
decreasing phosphodiesterase (PDE) and ACC activities, respectively, key proteins
regulated by insulin.
A. Kennedy et al. / Journal of Nutritional Biochemistry 21 (2010) 171–179
10,12-CLA alone [50,72,73,100]. PPARγ is a major activator of many
lipogenic genes, including glycerol-3-phosphate dehydrogenase, LPL
and lipin, as well as genes encoding lipid-droplet-associated
proteins such as perilipin, adipocyte-differentiation-related protein,
cell-death-inducing DFFA-like effector c and S3-12 . Thus,
10,12-CLA may exert its antilipogenic effects, in part, through its
ability to inhibit PPARγ activity. CLA repression of the lipogenic
transcription factor SREBP-1 and its target genes may also play an
important role. Finally, CLA suppression of insulin signaling may also
affect the activation or abundance of a number of lipogenic proteins,
including LPL, ACC, FAS, SCD1 and the insulin-dependent glucose
transporter 4 (GLUT4).
Interestingly, 10,12-CLA or a CLA mixture reduce the levels of
monounsaturated fatty acids in rodents [27,51] and primary human
adipocyte cultures . This may be due to the ability of CLA to
repress SCD1 expression  and function required for monosatu-
rated fatty acid synthesis [102,103]. However, 10,12-CLA supplemen-
tation reduced body weight in SCD1 knockout mice, simultaneously
increasing the ratio of 16:0/16:1fatty acids anddecreasing the ratio of
18:0/18:1 fatty acids . These results suggest that the antiobesity
properties of CLA may also rely on other desaturases, which may
include the isoenzyme SCD2. For instance, body fat loss in mice fed a
CLA mixture requires Δ6-desaturase .
2.4.2. CLA causes insulin resistance
Insulin-stimulated glucose uptake in WAT is mediated via GLUT4
(Fig. 4). Defect in insulin signaling or suppression of GLUT4
translocation to the plasma membrane is a primary cause of insulin
resistance in adipocytes (Figs. 4 and 5). Insulin resistance has been
reported in overweight or obese mice  or humans [92,105–107]
and in cultures of 3T3-L1  or human adipocytes [72,86,87]
following supplementation with a CLA mixture or 10,12-CLA alone.
Moreover, supplementation with a CLA mixture or 10,12-CLA has
been shown to induce hyperinsulinemia, which is associated with
insulin resistance in animals and humans (reviewed in Wang and
Jones ). CLA may inhibit insulin signaling by (a) activating
inflammatory pathways and stress kinases and (b) down-regulating
the expression of genes involved in the insulin signaling and glucose
In addition, some studies on 3T3-L1 cells  and cultures of
newly differentiated human adipocytes  have suggested that CLA
inhibits insulin signaling via increased expression of suppressor of
cytokine signaling 3 (SOCS-3). SOCS-3 impairs insulin signaling and
glucose uptake by promoting the phosphorylation of inhibitory serine
307 on insulin receptor substrate 1 (IRS-1), leading to its ubiquitina-
tion and proteasome degradation . CLA appears to induce SOCS-
3 indirectly via inflammatory cytokines such as TNFα and IL-6
[73,109,110]. 10,12-CLA treatment has also been demonstrated to
decrease the protein levels of insulin receptor β (IRβ)  and IRS-1
[24,86] — signaling proteins critical for insulin sensitivity. In addition,
10,12-CLA treatment reduced tyrosine phosphorylation (i.e., activa-
tion) of IRβ and IRS-1 in 3T3-L1 adipocytes .
10,12-CLA may directly impair the uptake of glucose and fructose
by suppressing the expression of their transporters. 10,12-CLA
decreased GLUT4 gene and protein expression [71,73,86] in cultures
of newly differentiated human adipocytes. Similarly, CLA reduced the
gene expression of GLUT4 and the glucose/fructose transporter
SLC2A5 in WAT and 3T3-L1 adipocytes supplemented with 10,12-
CLA may also cause insulin resistance via its effects on the insulin-
sensitizing hormone adiponectin. Adiponectin mRNA levels were
decreased following supplementation with 10,12-CLA in mice 
and in cultures of human adipocytes . Consistent with these data,
10,12-CLA or a CLA mixture decreased adiponectin assembly or
secretion in cultures of murine adipocytes, respectively [18,111].
Because adiponectin is a target gene of PPARγ , its suppression
may be due, in part, to 10,12-CLA-antagonizing PPARγ activity.
Accordingly, the PPARγ agonist rosiglitazone was able to prevent
CLA-induced suppression of adiponectin serum levels and insulin
resistance in mice . However, another PPARγ agonist, troglita-
zone, did not prevent the 10,12-CLA suppression of adiponectin
expression, although it prevented 10,12-CLA suppression of TG levels
and adiponectin oligomer assembly in 3T3-L1 adipocytes . These
results indicate that CLA may suppress adiponectin expression by a
Fig. 6. 10,12-CLA increases apoptotic cell death of (pre)adipocytes by increasing ER
stress. 10,12 activates upstream signals that induce cell stress, including ER stress and
ISR. These stress responses increase the levels of intracellular calcium, reactive oxygen
species and proteins that together induce apoptosis.
Fig. 7. Working model by which 10,12-CLA causes insulin resistance and delipidation in
adipocytes. We propose that 10,12-CLA induces upstream signals that cause (a) an ISR
that increases apoptosis, FFA release and inflammatory gene expression; (b) NFκB and
ERK activation that antagonizes PPARγ activity; and (c) increased UCP and lipolysis,
further enhancing FFA levels. Together, these CLA-mediated signals cause adipocyte
insulin resistance and delipidation.
A. Kennedy et al. / Journal of Nutritional Biochemistry 21 (2010) 171–179
2.4.3. CLA stimulates lipolysis
Lipolysis is the process by which stored TG is mobilized, releasing
free fatty acids (FFAs) and glycerol through the action of hormone-
sensitive lipase (HSL). Typically, when energy demand is increased,
lipolysis is up-regulated via cAMP-mediated signaling. CLA may
induce lipolysis in WAT through its activation of proinflammatory
pathways, thereby liberating FFA for uptake in metabolically active
tissues (i.e., liver and muscle) (Fig. 5). Acute treatment with mixed
CLA isomers or 10,12-CLA alone increased lipolysis in 3T3-L1
[19,100,113] and newly differentiated human adipocytes .
Furthermore, LaRosa et al.  observed increased mRNA levels of
HSL in C57BL/6J mice fed 10,12-CLA for 3 days; however, HSL levels
subsequently decreased following chronic (17-day) treatment.
Numerous studies in other species have investigated the effect of
long-term CLA supplementation on lipolysis. Studies with mice or
hamsters have demonstrated that chronic supplementation with a
mixture of CLA has no effect on lipolysis [115–117]. In contrast,
chronic treatment with CLA (1–200 μmol/L mixed isomers) reduced
glycerol release from isolated rat adipocytes . Consistent with
these data, FFA levels have been reported to be lower in the serum of
OLETF rats supplemented with a CLA mixture [1.0% (wt/wt) for 4
weeks] compared to controls . The lack of a chronic lipolysis
effect may be due to depleted TG stores in WAT, which can lead to
ectopic lipid accumulation seen in lipodystrophy syndromes. For
example, supplementation with a CLA mixture or 10,12-CLA alone
increased lipid accumulation in the liver of mice [119,120] and
hamsters [121,122]. In summary, CLA induces inflammatory adipo-
kines that likely impair insulin signaling, thereby decreasing TG
synthesis and increasing lipolysis, leading to decreased WAT mass
2.5. CLA regulation of apoptosis
2.5.1. CLA induces (pre)adipocyte apoptosis
Apoptosis is another mechanism by which CLA may be able to
reduce BFM. Studies using mice [33,34,50] or 3T3-L1 murine
adipocytes [52,113] supplemented with 10,12-CLA or a CLA mixture
have reported apoptosis in adipocytes. For example, mice fed a high-
fat diet containing a 1.5% (wt/wt) CLA mixture had an increased ratio
of BAX relative to Bcl2, inducer and suppressor of apoptosis in the
mitochondrial apoptotic pathway, respectively . Furthermore,
supplementation of C57BL/6J mice with a 1% (wt/wt) CLA mixture
reduced BFM and increased apoptosis and TNFα gene expression in
WAT . TNFα gene expression and secretion have also been
reported to be induced in mice by 10,12-CLA alone [16,24]. TNFα is a
potent inducer of apoptosis  and plays a critical role in adipocyte
function . TNFα gene expression was likewise induced by 10,12-
CLA in cultures of newly differentiated human adipocytes, although
its secretion was not detected [73,91].
Besides the TNFα/death receptor and mitochondrial pathways,
apoptosis can occur via activation of integrated stress response (ISR)
(Fig. 6). Microarray analysis revealed that 10,12-CLA treatment of
mice [1% (wt/wt)] and 3T3-L1 adipocytes (100 μmol/L) increased the
mRNA levels of genes involved in ISR such as activating transcription
factor 3, C/EBP homologous protein (CHOP), pseudokinase Tribbles 3/
SKIP 3 (TRIB3), X-box binding protein, and growth arrest and DNA
damage inducible protein (GADD34) . CHOP is known to possess
apoptotic characteristics, and activation of this protein can lead to
induction of GADD34 and TRIB3 [126,127]. Notably, CLA-induced ISR
activation in adipocytes was preceded by the induction of inflamma-
tory genes such as IL-6 and IL-8 . In mouse mammary tumor cells,
10,12-CLA treatment (20–40 μmol/L) increased CHOP expression and
endoplasmic reticulum (ER) stress, leading to apoptosis [128,129].
Collectively, these data suggest that CLA may induce adipocyte
apoptosis via ER stress and ISR, depending on the dose and isomer
used. In vivo studies are needed to investigate whether the apoptotic
effects of CLA on humans are specific for WAT.
3. Conclusion and implications
Supplementation with a mixture of CLA isomers or 10,12-CLA
alone reduces adiposity consistently in animal models, especially in
rodents, but reduces adiposity in only some human studies. Potential
reasons forthese species differencesinclude(a) theCLA isomersused,
(b) the dosage administered, and (c) the age, body weight, body fat or
metabolic status of the animals or subjects. Of the major isomers, only
10,12-CLA reduces the adiposity or TG content of WAT. Dosage
differences among species can be considerable; rodent studies
generally use ∼20 times more CLA per kilogram of body weight
than human studies.
Potential mechanisms responsible for these antiobesity properties
of 10,12-CLA include (a) decreasing energy intake by suppressing
appetite; (b) increasing energy expenditure in WAT, muscle and liver
tissue, or LBM; (c) decreasing lipogenesis or adipogenesis; (d)
increasing lipolysis or delipidation; and (e) apoptosis via adipocyte
stress, inflammation, and/or insulin resistance.
Based on these data, we propose the following working model
(Fig. 7) depicting the mechanisms by which 10,12-CLA decreases
WAT mass. We speculate that 10,12-CLA binds to a cell surface fatty
acid receptor or diffuses/flip-flops into adipocytes, thereby activating
upstream signals. These upstream signals induce ISR, FFA release, and
activation of NFκB and MAPKs that may directly antagonize PPARγ
activity. Increased release of PGs and cytokines may further
antagonize PPARγ activity, leading to insulin resistance and delipida-
tion. The resulting FFA accumulation in blood, liver and muscle
increases FFA oxidation, and FFA-induced insulin resistance in these
tissues. If energy expenditure is not sufficient to completely oxidize
these elevated levels of FFAs, hyperlipidemia, hyperglycemia, and
lipodystrophy can result.
Future studies are needed to identify potential upstream media-
tors of this proposed stress cascade in adipocytes. Elucidating these
mechanisms will provide valuable information on the efficacy,
specificity and potential side effects of CLA isomers as dietary
strategiesfor weightloss or maintenance. Suchknowledgeis essential
for the effective and safe use of CLA supplements to control obesity.
This work was supported by NIH R01 DK063070-07 (to M.M. and
S.M.), NIH F31DK076208 and a United Negro College Fund—Merck
predoctoral fellowship (to A.K.).
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