The biologically active isomers of conjugated linoleic acid
Michael W. Parizaa,*, Yeonhwa Parka, Mark E. Cooka,b
aDepartment of Food Microbiology and Toxicology, Food Research Institute,
University of Wisconsin-Madison, Madison, WI 53706, USA
bDepartment of Animal Sciences, Food Research Institute,
University of Wisconsin-Madison, Madison, WI 53706, USA
Numerous physiological effects are attributed to conjugated linoleic acid (CLA). The purpose of this
presentation is to consider these effects with respect to the cis-9,trans-11 and trans-10,cis-12 CLA isomers.
We review previously published data and present new findings that relate to underlying biochemical
mechanisms of action. Both isomers are natural products. The cis-9,trans-11 isomer is the principal dietary
form of CLA, but the concentrations of this isomer and the trans-10,cis-12 isomer in dairy products or beef
vary depending on the diet fed to cows or steers, respectively. The trans-10,cis-12 CLA isomer exerts spe-
cific effects on adipocytes, in particular reducing the uptake of lipid by inhibiting the activities of lipopro-
tein lipase and stearoyl–CoA desaturase. The trans-10,cis-12 CLA isomer also affects lipid metabolism in
cultured Hep-G2 human liver cells, whereas both the cis-9,trans-11 and trans-10,cis-12 CLA isomers
appear to be active in inhibiting carcinogenesis in animal models. We present new findings indicating that
the cis-9,trans-11 CLA isomer enhances growth and probably feed efficiency in young rodents. Accord-
ingly, the effects of CLA on body composition (induced by trans-10,cis-12 CLA) and growth/feed efficiency
(induced by cis-9,trans-11 CLA) appear to be due to separate biochemical mechanisms. We also show that
a 19-carbon CLA cognate (conjugated nonadecadienoic acid, CNA) inhibits lipoprotein lipase activity as
effectively as CLA in cultured 3T3-L1 adipocytes. Presumably, CNA is metabolized differently than the 18-
carbon CLA isomers, so this finding indicates direct activity of the administered compound as opposed to
acting via a metabolite. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Adipocytes; Skeletal muscle; Lipoprotein lipase; Stearoyl–CoA desaturase; 3T3-L1; Hep-G2; Carcinogenesis;
Feed efficiency; Growth; Body composition; Conjugated nonadecadienoic acid; Immune function; Cytokines; TNF-
alpha; IL-1; PPAR
0163-7827/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
Progress in Lipid Research 40 (2001) 283–298
* Corresponding author. Tel.: +1-608-263-6955; fax: +1-608-263-1114.
E-mail address: email@example.com (M.W. Pariza).
Numerous fatty acids with conjugated double bonds occur naturally in edible fats derived from
ruminant animals, for example milkfat and beef tallow [1–3]. Similar structures are generated
when linoleic acid is heated in base . The term conjugated linoleic acid and its acronym CLA
refer generically to this class of positional and geometric conjugated dienoic isomers of linoleic
acid, two of which (cis-9,trans-11 and trans-10,cis-12 CLA) are known to possess biological
activity (Fig. 1) .
Prior to 1987 scientific interest in CLA was confined largely to rumen microbiologists who
studied the cis-9,trans-11 CLA isomer as an intermediate in the biohydrogenation of linoleic acid
PPAR Peroxisome proliferator-activated receptor
SCD Stearoyl–CoA desaturase
TNFa Tumor necrosis factor-alpha
Conjugated linoleic acid
Conjugated nonadecadienoic acid
1. Introduction........................................................................................................................................................... 284
2. Formation of CLA isomers....................................................................................................................................286
2.2. Chemical synthesis.........................................................................................................................................287
3. Physiological effects................................................................................................................................................287
3.1. Body composition..........................................................................................................................................288
3.1.1.Effects on adipocytes.........................................................................................................................288
3.1.2. Effects on skeletal muscle..................................................................................................................292
3.1.3.Feed efficiency and growth................................................................................................................293
3.1.4.Effects on blood insulin.....................................................................................................................294
3.2. Hepatic lipid metabolism...............................................................................................................................294
3.4. Other effects...................................................................................................................................................295
4. Summary and conclusions......................................................................................................................................295
References ................................................................................................................................................................... 296
284 M.W. Pariza et al./Progress in Lipid Research 40 (2001) 283–298
[6,7]. This changed when Ha et al.  reported that CLA produced by base-catalyzed isomeriza-
tion of linoleic acid was an effective inhibitor of benzo(a)pyrene-initiated mouse epidermal neo-
plasia. Since then numerous biological and physiological effects of CLA have been reported [5,9]
(for a regularly updated listing of the scientific literature on CLA since 1987 see the Internet
Recently we reviewed evidence for CLAs multi-functionality and its implications regarding
possible biochemical mechanisms . Emerging evidence indicates that the cis-9,trans-11 and
trans-10,cis-12 CLA isomers produce different effects. Given the structural differences between
these isomers it is most unlikely that a single biochemical mechanism underlies these effects. In
fact, there is evidence indicating that more than one biochemical mechanism is involved in the
specific effects of the trans-10,cis-12 CLA isomer .
Fig. 1. Structures of trans-10,cis-12 CLA (upper panel),cis-9,trans-11 CLA (middle panel), and linoleic acid (lower
M.W. Pariza et al./Progress in Lipid Research 40 (2001) 283–298285
2. Formation of CLA isomers
Parodi  summarized the early literature describing the seasonal fluctuation of conjugated
dienes (now recognized as CLA) in cow’s milk. The amounts in spring and summer (when cows
were pastured) were substantially higher than in fall and winter (when cows were stall-fed). Par-
odi  established that cis-9,trans-11 CLA was the principal CLA isomer in milkfat, a finding
that has been confirmed [1–3].
Dhiman et al. [11–13] studied the effect of diet on the CLA content of cow’s milk. They
reported that certain high fat diets (particularly diets high in soybeans) substantially increased
milk CLA content, and reaffirmed that cis-9,trans-11 CLA was the principal isomer that was
modulated. The cis-9,trans-11 CLA isomer is produced in the rumen during the microbial bio-
hydrogenation of linoleic and linolenic acids [6,7] through an enzymatic conversion mechanism
that exhibits unusual properties . Some bacterial species in the large intestine of monogastric
animals also possess this capacity  but CLA formed in the large intestine would not be sub-
After formation in the rumen cis-9,trans-11 CLA may be directly absorbed or further meta-
bolized (biohydrogenated) by rumen microorganisms to trans-11-octadecenoic acid. Following its
absorption,trans-11-octadecenoic acid may then be converted by stearoyl–CoA desaturase (SCD)
within mammalian cells back to cis-9,trans-11 CLA [16,17]. This appears to be a major pathway
in the formation of cis-9,trans-11 CLA in cow’s milk [18,19].
Verhulst et al.  reported isolating a Propionibacter that converts linoleic acid to trans-
10,cis-12 CLA. Certain rumen bacteria (as yet unidentified) also appear to possess this capability
in that trans-10,cis-12 CLA has been observed in rumen digesta, as well as cis-9,trans-11 and
trans-9,trans-11 CLA . Cow’s milk is reported to contain trans-10,cis-12 CLA, as well as
trans-10-octadecenoic acid . By analogy to the formation of trans-11-octadecenoic acid in the
rumen via biohydrogenation of cis-9,trans-11 CLA,trans-10-octadecenoic acid may form in the
rumen via biohydrogenation of trans-10,cis-12 CLA . However, since mammals do not pos-
sess delta-12 desaturase, they could not then convert trans-10-octadecenoic acid back to trans-
10,cis-12 CLA. Accordingly, the trans-10,cis-12 CLA reported in ruminant tissues [18,22] would
seem to originate solely from trans-10,cis-12 CLA that is absorbed from the gastrointestinal tract.
The origins of other CLA isomers that have been reported to occur naturally in milkfat [1,2] are
not known but it is likely that they too result largely if not completely from bacterial metabolism
in the rumen.
Beef fat from steers fed control diet or the same diet supplemented with 2% or 4% soybean
oil, respectively, contained 0.1, 1.2, or 1%, respectively, of trans-10,cis-12 CLA . By contrast
the cis-9,trans-11 isomer content of the beef fat in these groups was unaffected (4, 4.1, or
4.6%, respectively) . This finding indicates that not just the absolute amounts but also the
ratios of CLA isomers produced in the rumen and subsequently found in tissue may be affected
Park and Pariza  reported that one lot of commercial horse sera contained
unexpectedly high levels of both cis-9,trans-11 CLA and trans-10,cis-12 CLA (0.53 and 0.4%
of total fatty acids, respectively). Since the horse has a hindgut fermentation area where
286 M.W. Pariza et al./Progress in Lipid Research 40 (2001) 283–298
long-chain fatty acid absorption is minimal, this finding appears to indicate that additional
research may be needed before we will fully understand the origins of CLA in mammalian
Dionisi et al.  found a number of CLA isomers in the membrane lipids of lactic
acid bacteria, but the origin of these was not determined (i.e. whether they were synthesized
de novo or were products of linoleic acid metabolism). Uchida  reported the apparent de novo
synthesis of unspecified CLA isomers by Pediococcus homari. Yang  isolated and charac-
terized a strain of Lactobacillus reuteri from rat colon that converts linoleic acid to cis-9,trans-11
2.2. Chemical synthesis
The goal of chemical synthesis should be to produce a fully characterized CLA com-
position with maximal biological activity. Accordingly, laboratory methods have been
developed to convert linoleic acid into CLA consisting mainly of the cis-9,trans-11 and trans-
10,cis-12 isomers. For example, CLA that we typically produce for experimental purposes
consists of the cis-9,trans-11 (40.8-41.1%),trans-10,cis-12 (43.5–44.9%), and trans-9,trans-11/
trans-10,trans-12 (4.6–10%) isomers [1,3,27]. It should be noted that some commercial CLA
preparations contain additional isomers with conjugated double bonds at the 8,10 or 11,13
positions . Reaney et al.  reviewed the literature on commercial production of CLA,
including discussion of reaction processes (raw materials, solvents, catalysts, reaction
vessels), reaction kinetics with regard to positional isomers, and product quality. There is
a considerable and growing body of knowledge on this topic which we will not further
review here, but rather refer readers to the CLA internet site listed above for additional
3. Physiological effects
Numerous seemingly beneficial physiological effects have been attributed to CLA including
inhibiting chemically induced carcinogenesis in several rodent models [8,29–31], enhancing the
immune response while reducing the catabolic effects of immune stimulation in rodents and
chickens [32,33], reducing atherosclerosis in rabbits  and hamsters , enhancing growth of
rats  and pigs , reducing body fat gain in mice [27,38–42], rats [43,44], hamsters , pigs
[46,47], dogs , and humans [49–51].
Most of these reports involved administering mixtures of CLA isomers that contained
mostly cis-9,trans-11 and trans-10,cis-12 CLA in approximately equal amounts, with other CLA
isomers at considerably lower levels [1,3,27]. However, it bears repeating that some commercial
CLA preparations contain additional isomers with conjugated double bonds at the 8,10 or 11,13
positions . The use of such CLA preparations in biological studies will complicate data
Emerging evidence indicates that the numerous biological effects of CLA are due to the sepa-
rate actions of the cis-9,trans-11 and trans-10,cis-12 isomers . It is also likely that some effects
are induced and/or enhanced by these isomers acting synergistically.
M.W. Pariza et al./Progress in Lipid Research 40 (2001) 283–298287
3.1. Body composition
The induction of changes in body composition is currently the most thoroughly studied of the
reported physiological effects of CLA.
Park et al.  were the first to report that dietary CLA could alter body composition. Control
diet or diet containing 0.5% CLA (cis-9,trans-11 and trans-10,cis-12 isomers in approximately a
50:50 ratio) were fed to weanling male or 6-week-old female mice for 4–5 weeks. At completion of
the study the CLA-fed animals displayed significantly reduced body fat coupled with enhanced
lean body mass relative to controls; however, the magnitude of the reduction in body fat relative
to controls was considerably greater than the magnitude of the enhancement in whole body pro-
tein. In addition the rate-limiting enzyme in beta-oxidation, carnitine palmitoyltransferase (CPT)
activity, was enhanced in skeletal muscle of CLA-fed animals.
In related in vitro experiments  it was observed that adding CLA to the media of 3T3-L1
adipocytes (at levels found in the blood of rats fed 0.5% CLA) significantly reduced lipoprotein
lipase (LPL) activity, which would in turn reduce fatty acid uptake. CLA-treatment also
enhanced the release of fatty acids from these cells into the media. This latter observation was
interpreted as indicating that CLA may enhance lipolysis, but more recent findings indicate that
this is instead a nonspecific phenomenon in 3T3-L1 adipocytes that is also produced by fatty
acids that have no effect on body composition in vivo [39, unpublished results].
Currently available in vivo and complementary in vitro findings pertaining to the effects of
CLA on body composition [27,38–57] should be considered in light of the interplay between
adipocytes (the principal site of fat storage) and skeletal muscle cells (the principal site of fat
3.1.1. Effects on adipocytes
A model for the effects of CLA on adipocytes is shown in Fig. 2. It appears conclusive
that trans-10,cis-12 CLA is the isomer that induces body composition change [39,45]. The
Fig. 2. A model for the effects of trans-10,cis-12 CLA on adipocytes and preadipocytes. For adipocytes, supporting
data are from studies with several species; for preadipocytes, supporting data are from studies with mice. See text for
288M.W. Pariza et al./Progress in Lipid Research 40 (2001) 283–298
preponderance of evidence indicates that this is due in part to a reduction in lipid uptake by
adipocytes [27,39,43,51,53,56,57]. The reduction in fatty acid uptake by adipocytes, in turn,
appears to be due to effects of CLA on SCD [52–55] and LPL [27,39] activities.
The mechanism whereby trans-10,cis-12 CLA reduces SCD activity appears to be two-pronged
in that both the expression [52,53] and enzymatic activity [54,55] of SCD are inhibited by trans-
10,cis-12 CLA. (However our latest data indicate that this two-pronged effect may not operate in
all species — Ntambi et al., unpublished). At present the data are insufficient to construct a model
for the inhibition of SCD expression by this isomer, but we have investigated the structural
requirements for inhibiting SCD activity .
The mechanism whereby trans-10,cis-12 CLA reduces LPL activity is unknown. However our
recent studies indicate that this effect is due to the direct action of the isomer rather than a
metabolite of the isomer. The basis for this conclusion is shown in Fig. 3. We prepared a 19-
carbon CLA cognate (conjugated nonadecadienoic acid, CNA) from nonadecadienoic acid, using
the same methodology that we utilize in producing CLA from linoleic acid . In cultured 3T3-L1
adipocytes, CNA inhibited lipoprotein lipase activity as effectively as CLA (Fig. 3). Presumably,
Fig. 3. Comparison of the inhibitory effects of CLA (
(&) on lipoprotein lipase (LPL) activity in cultured 3T3-L1 adipocytes. The IC50values for CLA and CNA, respec-
tively, were 50.3 and 40.9 mM. CLA was prepared as described  and the same method was used to prepare CNA from
nonadecadienoic acid (Nu-Chek Corp., Elysian, MN). Heparin-releasable LPL activity was determined according to
previously published methods . Data for CLA were taken from ref. .
), CNA (conjugated nonadecadienoic acid; *), and control
M.W. Pariza et al./Progress in Lipid Research 40 (2001) 283–298 289
CNA is metabolized differently than the 18-carbon CLA isomers. Accordingly we conclude
that the inhibition of LPL activity in this cell culture system is due to the administered
compound rather than a metabolite of the administered compound. Elsewhere we will report
these findings in more detail, including effects of CNA on other biochemical activities asso-
ciated with the trans-10,cis-12 and cis-9,trans-11 CLA isomers. It should be noted that these
findings with CNA (Fig. 3) do not preclude the possibility that CLA metabolites such as those
described by Sebedio et al.  might be involved in mediating other CLA-induced physiological
effects. The multiple effects of CLA may be the net result of several biochemical mechanisms that
involve both direct effects of the CLA isomers as well as C20:3 and C20:4 metabolites of the CLA
Tsuboyama-Kasaoka et al.  reported that apoptosis was induced in adipose tissue of mice
fed diet containing 1% CLA (isomer mixture, about 40% trans-10,cis-12 CLA), a finding that we
have confirmed (Xu and Pariza, unpublished). Trans-10,cis-12 CLA was also found to induce
apoptosis in cultured 3T3-L1 mouse adipocytes . By contrast, Azain et al.  reported that
feeding diet supplemented with 0.25 or 0.5% CLA for 5 weeks to Sprague–Dawley rats reduced
adipocyte cell size but not number. Hence the induction by CLA of apoptosis in adipose tissue
may be species dependent in that the effect was seen in the mouse  but not in the rat .
Based on available in vitro  and in vivo evidence [Xu and Pariza, unpublished] we conclude
that in mice CLA may induce apoptosis of preadipocytes (Fig. 2).
The possibility of effects of CLA (and specifically the trans-10,cis-12 isomer) on preadipocyte
differentiation remains unresolved. There is limited evidence indicating that CLA administration
might stimulate preadipocyte differentiation [59–62]. However this possibility was not supported
by in vitro [53,56] or in vivo  studies from other laboratories, where the data were actually
more consistent with CLA (and specifically the trans-10,cis-12 isomer) blocking, rather than
stimulating, preadipocyte differentiation.
At present there is little support for the hypothesis that CLA might enhance lipolysis [39,
unpublished results]. This conclusion, coupled with the additional information presented with the
model of Fig. 2, provides perspective for study design and data interpretation. In particular, one
would predict that CLA (specifically the trans-10,cis-12 isomer) would block body fat gain, but
not necessarily reduce body fat level which had accumulated prior to CLA administration.
For example, there is substantial evidence that CLA reduces body fat gain in young growing
animals [27,38–47]. However, there is little evidence for effectiveness in adult animals that are not
anabolic. Schoenherr and Jewell  found that dietary CLA failed to reduce body fat in adult
beagles, but was effective in blocking body fat gain in adult beagles that had been calorie
restricted but were then allowed free access to food that was supplemented with CLA. Similarly,
Zambell et al.  reported that CLA supplements did not reduce body fat in adult women,
whereas Atkinson  and Blankson et al.  provided evidence indicating that CLA supple-
ments were effective in reducing body fat gain in adult volunteers, a conclusion also supported by
in vitro studies with cultured human preadipocytes . These seemingly paradoxical results
would be expected if CLA reduces the uptake of lipid by adipocytes but has little or no effect on
lipolysis leading to fatty acid release from adipocytes (Fig. 2).
The model proposed in Fig. 2 does not preclude the possibility that under some circumstances
CLA might appear to reduce body fat that has already accumulated. If a major effect of trans-
10,cis-12 CLA is to block fat uptake into adipocytes without increasing lipolysis, then one might
290 M.W. Pariza et al./Progress in Lipid Research 40 (2001) 283–298
still expect body fat to decline in CLA-fed animals in proportion to the normal rate of lipolysis
(i.e. body fat turnover) in the species under study. For example, the data of Table 1 were obtained
by feeding control diet or CLA-supplemented diet to mature mice (i.e. ‘‘retired breeders’’). At the
conclusion of the study the mice fed CLA-supplemented diet exhibited significantly reduced body
fat relative to controls. Since we have no evidence that CLA enhances lipolysis in this species, it
seems most likely that the apparent CLA-induced reduction in body fat (Table 1) may be the
result of normal body fat turnover in this species (which may be relatively high compared with
larger mammals) coupled with an inhibition of fat uptake into adipocytes due to CLA adminis-
tration. One may speculate that a similar result was not seen in dogs  or humans [49,62] pos-
sibly because in these species the rate of fat turnover exceeded the experimental period. More
research is indicated to test this hypothesis.
A more complete picture of the molecular mechanism whereby the trans-10,cis-12 CLA isomer
induces these effects on adipocytes yet eludes us. The data of Fig. 3 support the conclusion that
the isomer acts directly on SCD and perhaps other key biochemical activities, but CLA isomer
metabolites  may also have a role in the overall effects of CLA on body fat regulation. It is
likely that the Peroxisome Proliferator-Activated Receptors (PPARs) are involved in some way in
the molecular mechanisms of action of both trans-10,cis-12 and cis-9,trans-11 CLA  but it is
also likely that we do not yet understand these interactions. For example, dietary CLA reduced
body fat equally well in PPAR-alpha null mice and the wild-type controls indicating that
this PPAR  is not involved in the mechanism of body fat reduction by trans-10,cis-12
Future refinements to the model of Fig. 2 should include consideration of the possibility that
adipocytes may not all respond similarly to molecular signals. Adipocytes are not confined to a
specific tissue, and evidence is emerging that the location of a given adipocyte within the body
may be crucial in determining how that adipocyte functions . Hence, one should not assume
that CLA (and trans-10,cis-12 CLA) will necessarily exhibit a single defined set of effects on all
adipocytes irrespective of other biological considerations. Rather, it is possible that the effects of
CLA on a given adipocyte will depend in part on the location, microenvironment and physi-
ological function of that adipocyte. Evidence for this possibility may already exist in that in pigs
CLA was reported ito reduce subcutaneous fat but enhance intramuscular fat . In addition,
whereas Azain et al.  found that dietary CLA reduced adipocyte cell number but not size in
Effect of feeding CLA-supplemented diet to mature (‘‘retired breeder’’) micea
Group Body weight (g) Food intake (g/mouse)Perimetric fat pad weight (g) % Body fat
aFemale ICR ‘‘retired breeder’’ mice (Harlan Sprague–Dawley, Madison, WI) were housed and acclimated as
described . The mice were then assigned to two groups and fed diet (TD 94060) supplemented with 5.5% corn oil
(control) or 5.23% corn oil plus 0.27% trans-10,cis-12 CLA for 12 days.
bSignificantly different, P<0.05.
n=6 for control, n=5 for test.
M.W. Pariza et al./Progress in Lipid Research 40 (2001) 283–298 291
growing female Sprague–Dawley rats fed a 7% fat (w/w) diet, Buison et al.  reported that
CLA supplementation failed to prevent body fat regain in obese mature weight-reduced female
Wistar rats fed a 40% fat (w/w) diet. Hence the CLA-induced reduction in body fat gain (Fig. 2)
may under some circumstances be overwhelmed by a diet that is excessively high in fat. More
research on these matters is needed.
3.1.2. Effects on skeletal muscle
The possible effects of CLA on skeletal muscle are more poorly understood than the effects of
CLA on adipocytes; we are in fact obliged to use the term ‘‘possible effects’’ because so little is
known. Fig. 4 shows a model for these possible effects.
The evidence for a specific effect of CLA on skeletal muscle is limited to the report that muscle
CPT activity was enhanced in skeletal muscle of CLA-fed mice  which may in turn lead to
enhanced beta-oxidation. This possibility has not been rigorously tested but there is evidence that
feeding CLA to mice enhances metabolic rate  and total energy expenditure without increas-
ing uncoupling gene expression . The extent to which these findings and conclusions extend to
other species has yet to be rigorously determined.
There is evidence indicating that dietary CLA may enhance whole body protein accretion in
mice . In rats [43,44], pigs [46,47] and possibly humans , CLA also appears to enhance lean
body mass gain relative to fat mass gain. The apparent anabolic effect in mice is small but in our
experience reproducible and statistically significant. For example, the pooled results from 10
independent experiments with growing mice conducted according to methods previously descri-
bed  indicate that whole body protein gain for controls was 5.208?0.093 g (n=81) and for
CLA-fed mice 5.366?0.099 g (n=85; P=0.041).
We  have proposed that muscle mass may be preserved or enhanced as a result of CLA-
induced changes in the regulation and/or action of Tumor Necrosis Factor-alpha (TNF-alpha)
and Interleukin-1 (IL-1), cytokines that profoundly affect skeletal muscle catabolism as well as
immune function. Accordingly, our proposal  indicates a possible physiological link between
the enhancement of lean body mass and immune function, and a biochemical mechanism that
may underlie such a link.
Fig. 4. Model for the possible effects of CLA on skeletal muscle cells. See text for details.
292 M.W. Pariza et al./Progress in Lipid Research 40 (2001) 283–298
There is limited evidence in mice indicating that a relative increase in lean body mass may
precede the reduction in body fat mass . This should be studied further, especially the
possibility that an increase in lean body mass may be causally related to a subsequent
reduction in body fat gain. Such a determination would be most important in that fat free
mass is conserved in direct proportion to food intake, e.g. more lean is conserved as more
food is consumed . One might speculate that CLA could be most effective in reducing body
fat gain under conditions of ad libitum feeding, where fat free mass would be maintained or
enhanced, rather than conditions of food restriction, where fat free mass might not be main-
tained. Of course more research is indicated to test this speculative and seemingly paradoxical
3.1.3. Feed efficiency and growth
Chin et al.  fed diets containing 0.5% CLA (prepared according to our published
methodology — ref. ) to primigravid female Fischer rats during gestation and lactation. At 10
days of lactation and thereafter, pups from the dams fed CLA exhibited significantly greater body
weight and (following weaning) improved feed efficiency relative to control animals. It has been
assumed that these effects were causally related to the changes in body composition (reduced
body fat, enhanced lean body mass) that are induced by CLA in many species [27, 38–57]. How-
ever, it now appears that this conclusion is not valid (Table 2) .
The data shown in Table 2 indicate that the cis-9,trans-11 isomer was active in enhancing body
weight gain and appeared also to enhance feed efficiency in weanling mice (with more animals this
Independent effects of dietary cis-9,trans-11 CLA or trans-10,cis-12 CLA on growth, feed conversion and body fat
levels in micea
(g/100 g diet)
(g/100 g diet)
body weight gain
0–3 week feed conversion
Percent body fat
00 9.9 0.12514.0
aWeanling ICR mice (6/group) (Harlan Sprague–Dawley, Madison, WI) were fed control diet, diet supplemented
with cis-9,trans-11 CLA (Matreya Inc., Pleasant Gap, PA), or diet supplemented with trans-10,cis-12 CLA (Natural
Lipids LTD, Hovdebygda, Norway). Data were analyzed using the general linear method of the statistical analysis
system using type IV sum of squares; planned relevant contrasts were tested to determine if feeding either isomer dif-
fered from control for the measured end points. From ref. .
bThe trend in body weight gain for the groups fed cis-9,trans-11 was significantly different from the control
cThe percent body fat was significantly reduced relative to control in the groups fed trans-10,cis-12 CLA
M.W. Pariza et al./Progress in Lipid Research 40 (2001) 283–298293
apparent effect may have become statistically significant). However, the cis-9,trans-11 isomer had
no effect on body fat levels. By contrast, the trans-10,cis-12 CLA isomer reduced body fat levels
relative to control but did not enhance either body growth or feed efficiency (it may even have
reduced feed efficiency — Table 2). Hence the overall effects of CLA on growth, feed efficiency
and body fat level appear to be due to the interaction of the two biologically active isomers.
Moreover, to achieve optimal effects on growth, feed efficiency, and body composition in young
growing animals, it appears necessary to feed a mixture containing both the cis-9,trans-11 and
trans-10,cis-12 CLA isomers .
It is important now to determine the biochemical basis for this intriguing apparent synergy
between the CLA isomers.
3.1.4. Effects on blood insulin
The possible effects of the CLA isomers on blood insulin is confused by marked differences in
species response. Mice fed CLA-supplemented diets developed mild insulin resistance [40,42]
which may be related to a shift toward enhanced use of fatty acids as fuel. By contrast, in the
Zucker diabetic fatty fa/fa rat, dietary CLA restored insulin sensitivity, a finding that led to the
proposal that CLA might be useful in treating type-2 diabetes . It is not yet known if these
paradoxical effects are due to the same isomer, nor is it yet possible to reconcile these findings
with respect to possible underlying biochemical mechanism(s).
Elsewhere we will report that in our 6 month study of 71 obese patients, plasma insulin and
glucose levels were unchanged in the group receiving CLA supplements (3 g/day) compared to
controls receiving vegetable oil placebo [Atkinson et al., unpublished]. Accordingly, it is possible
that neither of the effects of CLA on insulin sensitivity seen in mice [40,42] versus rats  may
apply to humans ingesting CLA supplements at 3 g/day.
3.2. Hepatic lipid metabolism
CLA exhibits several effects on hepatic lipid metabolism. Lee  and Pariza and Lee 
established that CLA (equal mixture of trans-10,cis-12 and cis-9,trans-11 isomers) reduced apoli-
poprotein B secretion in cultured human hepatoma Hep-G2 cells. Yotsumoto et al.  reported
that this effect was induced by the trans-10,cis-12 CLA isomer. (We will report elsewhere that the
key structural feature in inducing this effect is the trans-10 double bond — Storkson and Pariza,
unpublished.) The trans-10,cis-12 CLA isomer also inhibits the expression  and activity [53,54]
of hepatic SCD. This finding may relate to reports [73,74] indicating that the trans-10,cis-12 CLA
reduced triacylglyceride secretion in Hep-G2 cells although the effect of this isomer on triacyl-
glyceride synthesis in this cell line was less clear.
There is substantial documentation for species differences with regard to CLA-induced lipid
accumulation in the liver. Mice fed CLA-supplemented diets develop enlarged livers apparently
due to lipid accumulation [42,75] whereas hamsters fed CLA-supplemented diets develop liver
hypertrophy (i.e. hepatocyte enlargement) which is not due to lipid accumulation . (Neither of
these effects are considered by themselves to be evidence of liver damage or intoxication .) By
contrast rats [43,77] or pigs  fed CLA-supplemented diets exhibited no evidence of lipid
accumulation in the liver or enhanced liver weight. It is clearly important to determine the
molecular basis for these differences in physiological response.
294 M.W. Pariza et al./Progress in Lipid Research 40 (2001) 283–298
The biological activity of CLA was discovered because of its inhibitory effects on chemically
induced epidermal carcinogenesis in mice  and subsequent research expanded this finding to
include other rodent carcinogenesis models [29–31]. The biochemical mechanism whereby CLA
inhibits carcinogenesis is not yet known, but may involve effects on the metabolism of linoleic
acid  and vitamin A . Further, like the effects of the individual CLA isomers on growth,
feed efficiency and body fat level (Table 2), the effects of the isomers on carcinogenesis appear to
be complementary. The cis-9,trans-11 CLA isomer by itself has been shown to effectively reduce
chemically induced rat mammary neoplasia . However, there is also evidence for a synergistic
interaction between cis-9,trans-11 and trans-10,cis-12 CLA in inhibiting carcinogenesis at this site
. It is possible that both isomers might affect linoleic acid metabolism is a manner that reduces
mammary carcinogenesis in the rat model , as well as exerting separate individual biochemical
3.4. Other effects
The immune system is also affected by CLA . The first reported effects were that CLA
enhanced certain immune responses while reducing the adverse effects of immune-mediated
catabolism [32,33]. More recently dietary CLA has been shown to increase immunoglobulin
production in rat spleen lymphocytes  and to reduce antigen-induced histamine and PGE2
release from sensitized guinea pig tracheae . The precise roles of the individual CLA isomers
in these effects are not yet known, but it has been reported that trans-10,cis-12 CLA increases
lymphocyte proliferation in vitro .
Trans-10,cis-12 CLA has also been reported to inhibit milk fat synthesis in dairy cows .
This effect appears to be due to inhibition of SCD and fatty acid synthesis.
4. Summary and conclusions
We have discussed many of the reported physiological effects of CLA with specific regard to
the separate and/or synergistic actions of the two known biologically active isomers,cis-9,trans-11
and trans-10,cis-12 CLA. Previously published data were reviewed and new findings that relate to
underlying biochemical mechanisms of action were presented.
Both the cis-9,trans-11 and trans-10,cis-12 CLA isomers occur naturally in food. The cis-
9,trans-11 isomer is the principal dietary form of CLA, but the concentrations of this isomer and
the trans-10,cis-12 isomer in dairy products or beef vary depending on the diet fed to cows/steers,
The trans-10,cis-12 CLA isomer exerts specific effects on adipocytes. A model is presented
in which this isomer acts primarily by reducing the uptake of lipid into adipocytes by inhibiting
the activities of adipocyte LPL and SCD. The trans-10,cis-12 CLA isomer also affects lipid
metabolism in cultured Hep-G2 human liver cells and increases lymphocyte proliferation in
vitro, whereas both the cis-9,trans-11 and trans-10,cis-12 CLA isomers appear to be active in
inhibiting carcinogenesis in animal models. In addition we presented new findings indicating that
M.W. Pariza et al./Progress in Lipid Research 40 (2001) 283–298 295
the cis-9,trans-11 CLA isomer enhances feed efficiency and growth in young rodents. Accordingly
the effects of CLA on body composition (induced by trans-10,cis-12 CLA) and growth/feed
efficiency (induced by cis-9,trans-11 CLA) appear to be due to separate biochemical mechanisms.
We provided evidence indicating that a 19-carbon CLA cognate (CNA) inhibits lipoprotein
lipase activity as effectively as CLA in cultured 3T3-L1 adipocytes. Presumably, CNA is meta-
bolized differently than the 18-carbon CLA isomers, so this finding indicates direct activity of the
administered compound as opposed to acting via a metabolite. It is probable that some of the
effects of CLA are also due to CLA-induced changes in linoleic acid metabolism. It is likely, but
not yet proved that metabolites (e.g. C20:3, C20:4) of the CLA isomers are involved in some
aspects of the biochemical mechanism(s) whereby CLA induces its physiological effects. Given
the current pace of new discovery concerning CLA, it is likely that our current ideas and models
will change regularly and considerably over the next several years.
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