Food Chemistry 129 (2011) 21–27

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

The presence of the trans-10, cis-12 sequence does not have a bodyfat-lowering effect on jacaric acid, a conjugated linolenic acid isomer

Jonatan Miranda a, Alfredo Fernández-Quintela a, Itziar Churruca a, Josune Ayo b,Cristina García-Marzo b, Renaud Dentin c, María Puy Portillo a,⇑a Dpt. Nutrition and Food Sciences, Faculty of Pharmacy, University of the Basque Country, Paseo de la Universidad, 7. 01006 Vitoria, Spainb Food Research Division, AZTI-Tecnalia, Astondo bidea, 609. Parque Tecnológico de Bizkaia, 48160 Derio, Spainc Département d’Endocrinologie, Métabolisme et Cancer, INSERM U567, UMR CNRS, Institut Cochin 22, rue Méchain, 75014 Paris, France

a r t i c l e i n f o

Article history:Received 26 March 2010Received in revised form 30 November 2010Accepted 12 January 2011Available online 15 January 2011

Keywords:Jacaric acidBody fatLipoprotein lipaseAcyl-CoA oxidaseCarnitine palmitoyltransferase-IaPPARaPPARcRat

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.01.037

Abbreviations: ACC, acetyl-CoA carboxylase; ACconjugated linolenic acid; CPT-Ia, carnitine palmitoyltrlipase; PPAR, peroxisome proliferator-activated recepliferated response element; WAT, white adipose tissu⇑ Corresponding author. Tel.: +34 945 013067; fax:

E-mail address: mariapuy.portillo@ehu.es (M.P. Po

a b s t r a c t

The term conjugated linolenic acid (CLNA) includes multiple positional and geometric cis and trans iso-mers. The aim was to determine the potential effectiveness of jacaric acid, a CLNA isomer, on body fatreduction in rats fed a high-fat diet, and its ability to activate the peroxisome proliferator-activatedreceptors, PPARa and PPARc. CLNA did not modify adipose tissue weight nor did it modify the expressionof lipoprotein lipase, acyl-CoA oxidase and carnitine palmitoyltransferase-Ia, but it did reduce that ofPPARa and PPARc. Moreover, it was not able to activate these transcriptional factors. These results dem-onstrate that, despite the presence of the diene sequence trans-10, cis-12, in octadecatrienoic jacaric acid,it does not have body-fat-lowering properties, probably due to the first cis double bond placed in position8. Furthermore, unlike other CLNA isomers, such as punicic and a-eleostearic acids, jacaric acid does notactivate PPARa and PPARc.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction molecule for humans is still a matter for debate. In October 2010,

In recent years, many studies have been carried out on the bodyfat-lowering properties of the trans-10, cis-12 isomer of conjugatedlinoleic acid (CLA) (Bhattacharya, Banu, Rahman, Causey, & Fernan-des, 2006; Park & Pariza, 2009; Wang & Jones, 2004). Although thisfatty acid has a potent reducing effect on body fat accumulation inanimal models, its effects on humans are controversial. Namely, areduction in body fat has been found, in some of the publishedstudies performed in humans, while a similar number of papersfailed to find any decrease. Moreover, its effect on humans is farless prominent than that observed in rodents; in rodents, bodyfat reduction (induced by CLA) is quite strong (20–60%) whereas,in humans, this effect is rather weak (2–6%) (Navarro, Fernández-Quintela, Churruca, & Portillo, 2006; Salas-Salvado, Marquez-Sandoval, & Bullo, 2006; Terpstra, 2004; Whigham, Cook, &Atkinson, 2000). Consequently, the role of CLA as an anti-obesity

ll rights reserved.

O, acyl-CoA oxidase; CLNA,ansferase-Ia; LPL, lipoproteintor; PPRE, peroxisomal pro-

e.+34 945 013014.

rtillo).

EFSA (European Food Safety Authority) provided a scientific opin-ion addressing the substantiation of health claims in relation toCLA. On the basis of the data presented, the Panel concluded thata cause and effect relationship had not been established betweenthe consumption of CLA and reaching or maintaining normal bodyweight (EFSA Panel on Dietetic Products & Allergies, 2010a). Withregard to the relationship with body fat reduction, the Panel hasnot yet provided any scientific opinion.

Moreover, despite the beneficial effects of CLA described in ani-mal models in terms of its anti-obesity properties, and althoughEFSA has recently published a scientific opinion giving support tothe safety of CLA under specific conditions (EFSA Panel on DieteticProducts & Allergies, 2010b), some concerns over the potentialsafety of CLA still remain. This is the case for glucose intoleranceover 6 months of treatment or for diabetic patients, as well as foroxidative stress (Pariza, 2004; Park & Pariza, 2007).

Taking all that into account, a great deal of scientific workfocuses on researching new effective anti-obesity molecules with-out deleterious effects on health. Given this background, interest inconjugated linolenic acid (CLNA) isomers is growing.

CLNA is one of the more highly unsaturated forms of conjugatedacids, which includes multiple positional and geometric cis andtrans isomers. Unlike CLA, CLNA isomers are present at much

Table 1Lipid sources of experimental diets (g/kg diet).

Control CLNA

Palm oil 25.0 28.1Jacaranda mimosifolia seed oil – 15.8Sunflower oil 57.0 48.1Linseed oil 20.0 10.0Total 102 102

22 J. Miranda et al. / Food Chemistry 129 (2011) 21–27

higher levels in certain seed oils (Badami & Patil, 1980; Sevil, 2005;Takagi & Itabashi, 1982). Bitter gourd (Momordica charantia) oil andtung (Vernicia fordii) seed oil contain 60% and 70% of a-eleostearicacid (cis-9, trans-11, trans-13 18:3), respectively, pomegranate(Punica granatum) oil contains about 72% of punicic oil (cis-9,trans-11, cis-13 18:3), catalpa (Catalpa ovata) seed oil contains31% of catalpic acid (trans-9, trans-11, cis-13 18:3), pot marigold(Calendula officinalis) seed oil contains up to 60% of calendic acid(trans-8, trans-10, cis-12 18:3), and jacaranda (Jacaranda mimosifo-lia) seed oil contains 32% of jacaric acid (cis-8, trans-10, cis-12 18:3)(Arao et al., 2004; Koba et al., 2007).

Park et al. (2004) proposed that the trans-10, cis-12 conjugateddouble bond, in conjunction with a carboxyl group at C-1, isrequired for the inhibition of LPL activity, one of the most impor-tant mechanisms of action which explain the body fat-loweringeffect of CLA. According to this proposal, conjugated linolenic acidisomers maintaining this structure might be effective in reducingbody fat. However, Chardigny et al. (2003) demonstrated that cal-endic acid (trans-8, trans-10, cis-12), a CLNA isomer which sharesthe trans-10, cis-12 double bond with the effective CLA isomer,seems to be less effective than CLA. On the other hand, Saeboet al., 2005 showed that cis-6, trans-10, cis-12, an octadecatrienoicfatty acid not totally conjugated, which also shares the trans-10,cis-12 double bond, had no effect on milk fat synthesis in lactatingcows. Apparently, the third double bond, trans-8 or cis-6, reducesthe effect of the trans-10, cis-12 structure. Nevertheless, the poten-tial effectiveness of other CLNA isomers which share the trans-10,cis-12 structure with CLA, showing an additional double bond inanother position, cannot be discounted at this point.

As a result, the present study analysed the potential effective-ness of jacaranda seed oil, which is rich in jacaric acid, on bodyfat reduction in rats fed a high-fat diet, focusing on adipose tissuetriacylglycerol uptake and liver fatty acid oxidation. These are themost important metabolic pathways involved in the body fat-low-ering effect of other conjugated acids. Taking into account that ithas been demonstrated that other CLNA isomers are PPAR agonists(Chuang et al., 2006; Hontecillas, O’Shea, Einerhand, Diguardo, &Bassaganya-Riera, 2009), we sought to investigate, in an in vitrostudy, whether jacaric acid would modulate the activities of PPARaand PPARc, two transcriptional factors which control lipid metab-olism, shown to be involved in CLA effects.

Table 2Fatty acid composition of Jacaranda mimosifolia seed oil (% fatty acid methylesters of the total fatty acids).

Fatty acids %

16:0 (palmitic acid) 3.7 ± 0.0318:0 (stearic acid) 5.7 ± 0.0120:0 (eicosanoic acid) 0.5 ± 0.00cis-9 18:1 (oleic acid) 12.8 ± 0.06cis-9, cis 12 18:2 (linoleic acid) 41.6 ± 0.01cis-9, cis12, cis15 18:3 (linolenic acid) 0.4 ± 0.00trans-10, cis-12 18:2 0.7 ± 0.00cis-8, trans-10, cis-12 18:3 (jacaric acid) 32.8 ± 0.06trans-8, trans-10, cis-12 18:3 (a-calendic acid) 1.4 ± 0.02trans-8, trans-10, trans-12 18:3 (ß-calendic acid) 0.3 ± 0.02

Values are means ± SEM.

2. Materials and methods

2.1. Animals, diets and experimental design

The experiment was conducted with 16 male Wistar rats(140 ± 1 g) purchased from Harlan Ibérica (Barcelona, Spain) andits protocol was approved by the Animal Experimentation EthicsCommittee of the University of the Basque Country. Animals wereindividually housed in polycarbonate metabolic cages (TechniplastGazzada, Guguggiate, Italy), placed in an air-conditioned room(22 ± 2 �C) with a 12 h light–dark cycle. After a six days adaptationperiod, the rats were randomly divided into two groups of eightanimals, control group and CLNA. Both groups were fed for 7 weekswith semi-purified high-fat diets consisting of 409 g/kg of sucrose(local market), 200 g/kg of wheat starch (Vencasser, Bilbao, Spain),200 g/kg of casein (Sigma, St. Louis, MO, USA), 102 g/kg of fat (fordetails see Table 1), 30 g/kg of cellulose (Vencasser, Bilbao, Spain),4 g/kg of L-methionine (Sigma, St. Louis, MO, USA), and 4 g/kg ofcholine chlorhydrate (Sigma, St. Louis, MO, USA). Vitamin and min-eral mixes were formulated according to AIN-93G guidelines(Reeves, Nielsen, & Fahey, 1993) and supplied by ICN Pharmaceu-ticals (Costa Mesa, CA, USA). Jacaranda seed oil was extracted, induplicate, from the seeds of Jacaranda mimosifolia, as previously

described (Miranda et al., 2009). The lipid profile of jacaranda seedoil (Table 2) showed that jacaric acid represented 32.8% of the totalfatty acid methyl esters. Other CLNAs, a-calendic and b-calendic(1.4% and 0.3% respectively) were also detected. Jacaric acid wassupplemented at a level of 0.5% to the CLNA group. Consideringthat 0.5 is one of the most frequently used doses in CLA animalstudies (Li, Huang, & Xie, 2008), the same dose was selected inthe present work, in order to make it possible to compare theeffects of CLA and CLNA isomers. All animals had free access tofood and water.

At the end of the experimental period, animals were sacrificedunder anaesthesia (chloral hydrate) by cardiac exsanguination.White adipose tissue (WAT) from different anatomical locations(epididymal, perirenal and subcutaneous) and liver were dissected,weighed and immediately frozen at �80 �C.

2.2. Extraction and analysis of RNA and quantification by reversetranscription-polymerase chain reaction (RT-PCR)

Total RNA was isolated from 100 mg of epididymal adipose tis-sue and 100 mg of liver, using Trizol (Invitrogen, Carlsbad, CA,USA), according to the manufacturer’s instructions. RNA sampleswere then treated with DNA-free kit (Ambion, Applied Biosystems,USA) to remove any contamination with genomic DNA. The yieldand quality of the RNA were assessed by measuring absorbanceat 260, 270, 280 and 310 nm and by electrophoresis on 1.3% aga-rose gels. 1.5 lg of total RNA of each sample was reverse-tran-scribed to first-strand complementary DNA (cDNA) using aiScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA).

Relative lipoprotein lipase (LPL), acyl-CoA oxidase (ACO), carni-tine palmitoyltransferase-Ia (CPT-Ia) and PPARa and PPARc mRNAlevels were quantified, using real-time PCR with an iCycler™ -MyiQ™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA,USA). b-Actin mRNA levels were similarly measured and servedas the reference gene. 0.1 ll of each cDNA were added to the PCRreagent mixture, SYBR� Green Master Mix (Applied Biosystems,California, USA), with the upstream and downstream primers(300 nM each). Specific primers were synthesised commercially(Eurogentec, Seraing, Belgium), and the sequences are shown inTable 3.

Table 3Primers for PCR amplification of each gene studied SYBR� Green RT-PCR.

Primers Sense primer Antisense primer

ACO 50-ATC TCT GTG GTT GCT GTG GAG TCA-30 50-TCT GGA TGC TTC CTT CTC CAA GGT-30 (1)CPT-Ia 50-AAC TTT GTG CAG GCC ATG ATG 50-AGC TTG TGA GAA GCA CCA GCA -30 (2)PPARa 50-GAG AAA GCA AAA CTG AAA GCA GAG A-30 50-GAA GGG CGG GTT ATT GCT G-30

PPARc 50-ATTCTGGCCCACCAACTTCGG -30 50-TGGAAGCCTGATGCTTTATCCCCA -30

LPL 50-CAGCTGGGCCTAACTTTGAG-30 50-CCTCTCTGCAATCACACGAA-30

b-Actin 50-ACGAGGCCCAGAGCAAGAG-30 50-GGTGTGGTGCCAGATCTTCTC-30

ACO, acyl-CoA oxidase; CPT-IA, carnitine palmitoyltransferase-Ia; PPAR, peroxisome proliferator-activated receptor; LPL, lipoproteinlipase. (1) Ferreira et al., 2008. (2) (Rodríguez et al., 2008).

J. Miranda et al. / Food Chemistry 129 (2011) 21–27 23

The PCR parameters were as follows: initial 2 min at 50 �C,denaturation at 95 �C for 10 min, followed by 40 cycles of denatur-ation at 95 �C for 15 s, and combined annealing and extension at60 �C for 1 min, for PPARa, ACO and b-actin. In the case of LPLand PPARc, annealing was performed at 60.8 and 63.9 �C, respec-tively, for 30 s. CPT-Ia PCR was carried out with an initial 2 minat 50 �C, denaturation at 95 �C for 10 min, followed by 45 cyclesof denaturation at 95 �C for 15 s, annealing at 66.5 �C for 30 s andextension at 60 �C for 30 s.

All sample mRNA levels were normalised to the values of b-ac-tin and the results expressed as fold changes of threshold cycle (Ct)value relative to controls using the 2�DDCt method (Livak & Sch-mittgen, 2001).

2.3. Gas chromatography for fatty acid analysis

2.3.1. Lipid extractionDiet samples and frozen samples of 0.5–1 g of tissues (liver and

epididymal adipose) were kept at 0 �C in an ice bath, and homog-enised in trichloromethane:methanol, 2:1 (vol/vol), using a PotterHeidolph RZR 1 homogeniser (Schwabach, Germany). Sampleswere allowed to stand at room temperature for 1 h. After dryingunder nitrogen, the sample was dissolved in 1 ml of trichlorome-thane for methylation.

2.3.2. Methylation of TAGSodium methoxide (500 ll in 0.5 M methanol) (Sigma–Aldrich,

Steinheim, Germany) was added to lipid extracts to prepare thefatty acid methyl esters (FAME). The mixture was heated at 50 �Cfor 10 min, and the reaction was stopped with 100 ll of glacial ace-tic acid. Water (5 ml) was added, and the required esters wereextracted twice with n-hexane (5 ml). The hexane layer was driedover anhydrous sodium sulphate (Merck, Frankfurt, Germany) andfiltered with a #1 Whatman filter paper. The solvent was removedunder reduced pressure on a rotatory evaporator and dissolved in500 ll of n-heptane containing 50 ppm of BHT as stabiliser, beforethe GC analysis.

2.3.3. Analysis by GCSamples with FAME were transferred to GC vials and analysed

with a Hewlett Packard HP6890 gas chromatograph (Hewlett Pack-ard, Palo Alto, CA) equipped with a FID, split/splitless injectionport, an HP Chemstation software data system and an HP7673autosampler (Hewlett Packard). The analytical column was a fusedsilica capillary Chrompack CP-Sil 5 CB column (60 m � 0.25 mmi.d., 0.25 lm film thickness) from Varian, Inc (Palo Alto, CA). Theoven temperature was initially programmed at 40 �C (for 2 min)and raised to 175 �C at a 10 �C/min rate, then held for 27 minand then raised to 215 �C at a rate of 0.5 �C/min. Injection (1 ll)was run in split (50:1) mode. Helium was the carrier gas at con-stant flow (1 ml/min) and make-up gas for the FID. The injectorwas maintained at 200 �C and detector at 250 �C. FAME were

identified by comparison of retention times with standards (Sigma,St. Louis, MO). Pure CLA and CLNA isomers were purchased fromLarodan Fine Chemicals AB (Malmö, Sweden). FA with concentra-tions lower than 0.3 wt.% were considered minor, and not shownunless they were CLA or CLNA isomers.

2.4. Reporter activity assay

HEK cells (Human Kidney Embrionic cells)-293T cells weremaintained and transfections were carried out as previouslydescribed (Dentin et al., 2007). HEK-293T were transiently trans-fected with 25 ng of the reporter expression vector under study,peroxisome proliferator response element (PGL2-PPRE-Luc), andwith 25 ng of the internal control vector (b-galactosidase), usingLipofectamin 2000� (Invitrogen, Carlsbad, CA, USA), according tothe manufacturer’s instructions. Cells were divided into threeexperimental groups: cells in basal state (transfected with pBlue-scrip 5 ng), cells with over-expression of PPARa (transfected with5 ng of PPARa) and cells with over-expression of PPARc: (transfec-ted with 5 ng of PPARc). WY-14643 (a PPARa agonist) (Calbio-chem-Merk, Darmstadt, Germany) and rosigliatzone (a PPARcagonist) (Cayman Chemical, Michigan, USA) were used as positivecontrol of PPARa and PPARc receptors activation, respectively.After 24 h, these cells were treated with the jacaric acid at differentdoses (1, 5, 10, 25, 50 and 100 lM) for 18 h. CLNA extract, as wellas PPAR ligands, were dissolved in 95% ethanol. At this point, lucif-erase activity was measured. Luciferase activity was normalised fortransfection efficiency using a b-galactosidase reporter (RSV b-gal)as an internal control. Data are means of nine determinations withluciferase activity normalised to b-galactosidase activity, con-ducted in three different experiments.

b-Galactosidase assays for normalisation of PPRE luciferaseactivity were performed using 10 ll of cell lysate, 50 ll of 2� buf-fer (1.33 mg/ml 2-nitrophenyl b-D-galactopyranoside, 100 mM2-mercaptoethanol, 2 mM magnesium chloride, 200 mM sodiumphosphate, pH 7.5, all purchased from Sigma, St. Louis MO, USA)and 40 ll of water in each well of a clear 96-well plate. The platewas covered and incubated for 30 min at 37 �C, and absorbanceat 405 nm was determined with a Lumimark Plus (Bio-Rad,Hercules, CA, USA) plate reader. Lysate samples were assayed intriplicate. Lysates from untransfected cells were used as controlsfor background activity.

2.5. Statistical analysis

Results are presented as means ± standard error of the mean.Statistical analysis was performed using SPSS 16 0 (SPSS Inc. Chi-cago, IL, USA). Data were analysed by two-tailed Student’s t test(in vivo study) or by one-way analysis of variance with fixed factor‘‘jacaric acid concentration’’ at seven levels (0, 1, 5, 10, 25, 50 and100 lM), followed by the Tukey post hoc test (in vitro study). Statis-tical significance was set-up at the P < 0.05 level.

24 J. Miranda et al. / Food Chemistry 129 (2011) 21–27

3. Results

3.1. Body weight, food intake and white adipose tissue weights

No statistical differences in food intake or final body weightwere found between the two experimental groups. Daily intakesof 18:1, 18:2 and 18:3 fatty acids did not show significant differ-ences between the two experimental groups. Similarly, adiposetissue weight, from the different anatomical regions analysed

Table 4Body weight, food intake and adipose tissue weights in rats fed on the experimentaldiets for 7 weeks.

Controlgroup

CLNAgroup

Student0s ttest

Food intake (g/d) 17.4 ± 0.20 16.9 ± 0.40 NSFatty acid intakes (mg/d)14:0 (miristic acid) 9 ± 0.1 9 ± 0.2 NS16:0 (palmitic acid) 318 ± 3.4 305 ± 7.2 NS18:0 (stearic acid) 72 ± 0.8 69 ± 1.6 NScis-9 18:1 (oleic acid) 337 ± 3.6 323 ± 6.5 NScis-9,12 18:2 (linoleic acid) 866 ± 6.9 874 ± 20.5 NScis-9,12,15 18:3 (linolenic acid) 161 ± 1.7 84 ± 2.0 NScis-8, trans-10, cis-12 18:3 (jacaric

acid)0 75 ± 1.8 NS

Final body weight (g) 329 ± 6.1 346 ± 8.3 NSIBAT (mg) 920 ± 52.3 950 ± 70.1 NSWhite adipose tissue weights (g)Perirenal 10.4 ± 0.66 10.2 ± 0.82 NSEpididymal 9.3 ± 0.36 9.4 ± 0.59 NSSubcutaneous 12.7 ± 0.44 12.8 ± 1.29 NSR adipose tissues 32.6 ± 0.99 32.4 ± 2.12 NS

Values are means ± SEM; NS, not significant.

0

0.5

1

1.5

PPARα

mR

NA

Lev

els

(AU

)

**

0

0.5

1

1.5

2

CPT-Ia AC

mR

NA

Lev

els

(AU

)

Fig. 1. (A) PPARa and PPARc mRNA levels measured by real-time PCR, in liver and epididjacaric acid. Values are expressed relative to control group and results are given as me⁄P < 0.05. (B) CPT-Ia, ACO and LPL mRNA levels measured by real-time PCR, in liver and e0.5% of jacaric acid. Values are expressed relative to control group and results are given

(perirenal, epididymal and subcutaneous), were not modified inCLNA rats when compared with the controls (Table 4).

3.2. Enzyme and transcriptional factor expressions in tissues

mRNAs of PPARa in liver and PPARc in adipose tissue were sig-nificantly reduced, by �36% and �61%, respectively (P < 0.05 andP < 0.01) (Fig. 1A). Notwithstanding, CLNA feeding did not modify

PPARγ

Control

CLNA*

O LPL

Control

CLNA

ymal adipose tissue from rats fed on high-fat diets supplemented or not with 0.5% ofans with the standard errors of the means, shown by vertical bars. ⁄⁄P < 0.01 andpididymal adipose tissue from rats fed on high-fat diets supplemented or not withas means with the standard errors of the means, shown by vertical bars.

Table 5Fatty acid profile (% of total fat) of liver and adipose tissue of rats fed on a dietsupplemented or not with 0.5% jacaric acid for 6 weeks.

Liver Epididymal adiposetissue

Control Jacaricacid

Control Jacaricacid

14:0 0.7 ± 0.13 0.6 ± 0.07 1.3 ± 0.06 1.5 ± 0.0216:0 25.1 ± 0.80 24.3 ± 0.82 24.3 ± 0.79 25.9 ± 0.17cis-7 16:1 2.6 ± 0.32 3.2 ± 0.74 5.5 ± 0.48 7.0 ± 0.7418:0 18.5 ± 0.42 18.7 ± 0.82 3.1 ± 0.08 2.7 ± 0.16cis-9 18:1 12.5 ± 1.30 11.3 ± 0.94 29.6 ± 0.23 29.8 ± 0.16cis-7 18:1 3.6 ± 0.25 4.1 ± 0.34 2.6 ± 0.04 2.8 ± 0.18cis-9, cis-12 18:2 0.6 ± 0.08 0.3 ± 0.04 4.9 ± 0.23 2.1 ± 0.07cis-9, cis-12, cis-15

18:317.7 ± 0.53 14.9 ± 1.15 28.1 ± 1.06 25.7 ± 0.92

cis-5, cis-8, cis-11,cis-14 20:4

18.4 ± 1.29 21.8 ± 0.99 0.4 ± 0.02 0.3 ± 0.02

trans-10, cis-12 18:2 0.3 ± 0.14 0.7 ± 0.14 0.4 ± 0.06 2.0 ± 0.15trans-8, trans-10,

cis-12 18:3ND 0.1 ± 0.03 ND 0.1 ± 0.01

Values are means ± SEM; ND, Not detected.

Basal

PPARγ

PPARα

JACARIC ACID 1 μM

0

500000

1000000

1500000

2000000

2500000

3000000

CONTROL

***

JACARIC ACID 1μM

JACARICACID 5μM

JACARIC ACID 10μM

JACARIC ACID 25μM

JACARIC ACID 50μM

JACARIC ACID

100μM

ROSIGLITAZONE

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

CONTROL JACARIC

ACID 1μM

JACARIC

ACID 5μM

JACARIC

ACID 10μM

JACARIC

ACID 25μM

JACARIC

ACID 50μM

JACARIC ACID

100μM

WT-14643

**

Rel

ativ

e L

ucif

eras

e A

ctiv

ity

JACARIC ACID 10μM

JACARIC ACID 25μM

JACARIC ACID 50μM

JACARICACID

100μM

Rel

ativ

e L

ucif

eras

e A

ctiv

ity

0

100000

200000

300000

400000

500000

CONTROL ROSIGLI-TAZONE

WT-14643

*

JACARICACID 5μM

JACARIC ACID 1μM

Rel

ativ

e L

ucif

eras

e A

ctiv

ity

Fig. 2. Effects of jacaric acid on peroxisome proliferator response element (PPRE) reporter activity of HEK-293T cells at basal state, or cells with PPARc/PPARa receptors over-expressed. Values are means with the standard errors of the means, shown by vertical bars. Rosigliatzone and WY-14643 were used as positive control of PPARc and PPARareceptors activation, respectively. ⁄⁄⁄P < 0.001, ⁄⁄P < 0.01 and ⁄P < 0.05.

J. Miranda et al. / Food Chemistry 129 (2011) 21–27 25

the expression of the enzymes CPT-Ia, ACO or LPL, regulated bythese nuclear factors (Fig. 1B).

3.3. Fatty acid profile in adipose tissue and liver

Fatty acid profile, expressed as percentage of total fat, shows thatsmall amounts of jacaric acid are present in both tissues (0.08% inboth tissues). The amounts of trans-10, cis-12 found in these tissueswere 2.04% in adipose tissue and 0.71% in liver (Table 5).

3.4. Reporter activity assay

The absence of PPARc and PPARa activation, by increasing con-centrations of jacaric acid, is illustrated in Fig. 2. Treatment (18 h)with jacaranda oil at different doses (1, 5, 10, 25, 50 and 100 lM ofjacarid acid) did not modify luciferase activity of cells at basal state,or cells with PPARc/PPARa receptors over-expressed, showing thatjacaric acid is not able to activate these transcriptional factors.

4. Discussion

A great deal of work is being carried out by the scientific com-munity in order to identify and characterise new naturally occur-ring molecules which are orally active and safe, for the purposeof chronic disease prevention. In recent years, growing interesthas focussed on CLNA isomers. As far as we know, there are onlya few studies devoted to analysing the effects of these CLNA iso-mers on body fat accumulation so far: cis-9, trans-11, trans-13(Chuang et al., 2006), trans-9, trans-11, cis-13 (Hontecillas, Diguar-do, Duran, Orpi, & Bassaganya-Riera, 2008), cis-9, trans-11, cis-13(Arao et al., 2004; Hontecillas et al., 2009; Koba et al., 2007),trans-8, trans-10, cis-12 (Chardigny et al., 2003) or a non-identifiedCLNA mixture (Koba et al., 2002).

Several authors have indicated that the introduction of a dou-ble bond in the carbon chain between the trans double bondlocated at C10 and C11 and the carboxyl group negates or sub-stantially reduces the antilipogenic activity associated with thetrans-10, cis-12 conjugated bond system (Chardigny et al.,2003; Saebo et al., 2005). Nevertheless, this conclusion has been

26 J. Miranda et al. / Food Chemistry 129 (2011) 21–27

achieved by using only a real CLNA isomer (trans-8, trans-10, cis-12) and an octadecatrienoic isomer (cis-6, trans-10, cis-12). In or-der to shed more light on this issue, we considered it interestingto test a new CLNA isomer with, not only the trans-10, cis-12structure but also a double bond placed at C8 but in the cisconfiguration.

Rats fed the diet enriched with jacaranda seed oil did not showa reduction in adipose tissue weights. Considering the smallamount of other conjugated fatty acids present in jacaranda seedoil (Table 2), it may be said that the lack of body fat reduction ob-served in the present experiment does not result from a maskingeffect of these minority components. Consequently, it may be sug-gested that jacaric acid lacks anti-obesity properties, and so rea-sons to justify this should be sought.

Two important mechanisms involved in the body fat-loweringeffect of trans-10, cis-12 CLA were the inhibition of triacylglyceroluptake in adipose tissue, and the activation of fatty acid oxidationin the liver (Wahle, Heys, & Rotondo, 2004; Wang & Jones, 2004).This being so, in order to gain more insight into the reasons forthe lack of jacaranda oil feeding efficacy, we analysed the effectsof this oil on the expressions of LPL, CPT-Ia and ACO enzymes.These play a key role in these two metabolic pathways. The effecton the expression of the transcriptional factors which control theseenzymes, PPARc and PPARa, was also evaluated.

Rats fed the diet enriched with jacaranda oil showed decreasedexpressions of both PPARc and PPARa. However, this effect wasnot observed in the expressions of the analysed enzymes (LPL,CPT-Ia and ACO). This absence of effect is in line with the lack ofeffect on body fat, but it does not fit well with the decrease inthe expression of the transcriptional factors. In order to shed lighton this apparent contradiction, we carried out an in vitro experi-ment in order to determine whether jacaric acid was able to acti-vate PPARc and/or PPARa. Thus, if this CLNA isomer was anactivator of these transcriptional factors, their reduced expressioncould be a consequence of this fact, and a sort of compensation be-tween these two phenomena could be proposed.

It has been reported that the presence of diene, triene or tetr-aene systems in fatty acids is often an indication of biologicalactivity and that these suggest agonistic effects on nuclear recep-tor (Hontecillas et al., 2008). More specifically, the authors statethat these plant-derived conjugated triene fatty acids containsome chemical signatures consistently found in most naturallyoccurring agonists of PPARs. Published data concerning this issueonly refer to punicic acid (cis-9, trans-11, cis-13 CLNA) and a-ele-ostearic acid (cis-9, trans-11, trans-13 CLNA). Thus, in an in vitrostudy performed in 3T3-L1 adipocytes, Hontecillas et al. (2009)reported that punicic acid, included in the diet of mice, activatedboth PPARc and PPARa, leading to suppression of obesity-relatedinflammation and greater glucose tolerance. In another study,Chuang et al. (2006) found that a-eleostearic acid activatedPPARa in a transactivation assay performed with a clone ofCHOK1 cells.

In contrast with these studies, in the present work the reporterluciferase-based assay of PPAR transactivation showed that jacaricacid did not activate PPARc or PPARa. It is important to emphasisethat the triene structure of jacaric acid (cis-8, trans-10, cis-12) dif-fers importantly from that of the two CLNA isomers mentioned.The absence of PPAR activation indicates that our hypothesis thata lack of compensation between the reduction in PPARs expressionbrings about its activation is not valid.

It has been reported that CLA isomers have been detected in ser-um and tissues of animals fed CLNA isomers, suggesting that CLNAisomers could metabolise, to some extent, into CLA isomers. Thus,the effects of CLNA isomers can be, at least in part, a result of thederived CLA isomers (Koba et al., 2007; Tsuzuki et al., 2004; Yama-saki et al., 2006). On the other hand, several studies have

demonstrated, in rats, that CLA isomers are agonists of PPARa(Moya-Camarena, Van den Heuvel, & Belury, 1999a; Moya-Cama-rena, Vanden Heuvel, Blanchard, Leesnitzer, & Belury, 1999b) andPPARc (Belury, 2002; Houseknecht et al., 1998).

Taking this fact into consideration, it could be hypothesised thatsuch a metabolic shift also took place in our experiment, thus con-verting jacaric acid into the active form of CLA (trans-10, cis-12).Consequently, a compensation between the reduction in transcrip-tional factors expressions induced by jacaric acid, and their activa-tion by CLA-derived isomers, or even their corresponding delta-6desaturase metabolites, which are higher PPARs activators (Belury,2002), could have occurred.

In order to check this hypothesis, gas chromatography was usedto analyse the fatty acid profile in adipose tissue and liver. It wasfound that a small amount of jacaric acid was stored in both tis-sues. Trans-10, cis-12 was also present in tissue samples. Althoughthe diet provided to jacaranda-treated animals contained trans-10,cis-12 CLA, its percentage (0.74% in jacaranda oil and 0.012% in thediet) does not justify the amounts of this isomer found in both tis-sues (2.04% and 0.71% of total fatty acids in adipose tissue and li-ver, respectively). This conclusion is based on a previous studyfrom our group: when animals were fed on a diet containing0.5% of trans-10, cis-12, the percentages of this isomer were0.77% and 1.35% of total fatty acids in adipose tissue and liver,respectively (Zabala, Portillo, Macarulla, Rodríguez, & Fernández-Quintela, 2006). Thus, the present results support the hypothesisthat a part of the jacaric acid is converted into trans-10, cis-12 CLA.

5. Conclusions

All these results, as a whole, support the conclusion that theintroduction of a double bond in the carbon chain between thetrans double bound located at C10 and C11 and the carboxyl groupnegates or reduces the effectiveness of isomers containing thetrans-10, cis-12 conjugated bond system (Chardigny et al., 2003;Saebo et al., 2005), and this effect seems to be independent ofthe cis or trans configuration. Moreover, in contrast to other CLNAisomers, such as punicic and a-eleostearic acids, jacaric acid doesnot activate PPARa and PPARc.

Acknowledgements

J. Miranda is a recipient of a doctoral fellowship from the Min-isterio de Educación y Ciencia. Palm oil was a generous gift fromAgra-Unilever Foods España S.A. (Leioa, Spain). PPAR response ele-ment (PPRE) luciferase reporter plasmid (pPPRE-Luc), as well asPPARa and PPARc receptor expression plasmids, were kind giftsfrom Dr. Renaud Dentin.

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