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Isomer-specific regulation of differentiating pig preadipocytes by conjugated linoleic acids¹

T. D. Brandebourg^* and C. Y. Hu^,2

^* Department of Animal Sciences and and College of Agricultural Sciences, Oregon State University, Corvallis 97331

	Abstract

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Conjugated linoleic acids are a group of geometric and positional isomers of linoleic acid that decrease body fat in growing animals by a poorly understood mechanism. The objective of this study was to investigate the isomer-specific effect of CLA on the proliferation and differentiation of pig preadipocytes in primary culture. The effect of CLA on preadipocyte proliferation was determined using cleavage of the tetrazolium salt, WST-1, as a marker for proliferation. Preadipocyte number was decreased in a dose-dependent fashion by trans-12,cis-10 CLA (P < 0.05). No other fatty acid affected preadipocyte number. Differentiation was monitored on d 10 after induction morphologically, enzymatically, and by measuring the mRNA abundance of key adipogenic transcription factors. Both a crude CLA preparation containing a mixture of CLA isomers (CLA-mix) and the pure trans-10,cis-12 CLA isomer inhibited glycerol-3-phosphate dehydrogenase (GPDH) activity in a dose-dependent fashion, with trans-10,cis-12 CLA being more potent (P < 0.01) than the CLA-mix. Cis-9,trans-11 CLA failed to decrease GPDH activity; however, increasing concentrations of cis-9,trans-11 CLA tended to blunt the inhibitory effect of trans-10,cis-12 CLA on GPDH activity (P < 0.09), suggesting that cis-9,trans-11 CLA may antagonize the action of trans-10,cis-12 CLA in porcine adipocytes. Finally, the isomer-specific effect of CLA on adipogenic transcription factor gene expression was investigated. Trans-10,cis-12 CLA decreased expression of peroxisome proliferator-activated receptor (PPAR ; P < 0.01) and sterol regulatory element-binding protein-1c (SREBP-1c; P < 0.05) mRNA, while failing to alter the expression of CCAAT/enhancer binding protein (C/EBP) mRNA. Interestingly, both the CLA-mix and the trans-10,cis-12 CLA isomer increased the mRNA abundance of chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF; P < 0.002). No other fatty acid affected COUP-TF mRNA levels. Collectively these data support the concept that CLA decreases fat accretion in pigs, in part by inhibiting preadipocyte proliferation and differentiation, with trans-10,cis-12 CLA being an active isomer eliciting these effects. Furthermore, trans-10,cis-12 CLA inhibits porcine preadipocyte differentiation by a mechanism that involves the down-regulation of PPAR and SREBP-1c mRNA. This mechanism is independent of changes in C/EBP mRNA abundance and may involve COUP-TF.

Key Words: Adipogenesis • Chicken Ovalbumin Upstream Promoter Transcription Factor 1 • Conjugated Linoleic Acid • Primary Culture • Swine

	Introduction

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Conjugated linoleic acids are naturally occurring isomers of linoleic acid that are associated with antiatherogenic, antidiabetic, and antitumorigenic action in mammals (reviewed by Pariza et al., 2000; Brown and McIntosh, 2003). Several trials suggest that pigs fed CLA deposit less fat and have improved body composition (Dugan et al., 1997; Azain, 2003; Dugan, 2004). Thus, CLA holds great promise as a feed additive in swine diets.

Fat deposition in swine results from the additive contributions of an increase in adipocyte number and size. Although CLA can limit adipose tissue accretion in growing pigs, the underlying mechanisms are poorly understood. In vitro studies utilizing 3T3-L1 preadipocytes suggest that CLA may limit adipocyte number through inhibitory actions on both preadipocyte proliferation and differentiation (Brodie et al., 1999; Evans et al., 2001; Kang et al., 2003). Although hyperplasia of human primary preadipocytes is potently inhibited in vitro by the trans-10,cis-12 CLA isomer (Brown et al., 2003), it has been reported that CLA fails to inhibit the proliferation and differentiation of pig preadipocytes, and it may even stimulate the differentiation of these cells (Ding et al., 2000; McNeel and Mersmann 2003). Given that a stimulatory effect of CLA on adipogenesis seems at odds with the ability of CLA to decrease carcass adiposity, the effect of CLA on the hyperplasia of adipocytes in pigs remains controversial. Our objective was to further characterize the isomer-specific effect of CLA on the proliferation and differentiation of pig preadipocytes in primary culture.

	Materials and Methods

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Materials
Linoleic acid was purchased from Sigma-Aldrich (St. Louis, MO). The 95% CLA mixture (CLA-mix; 40% cis-9,trans-11 CLA; 44% trans-10,cis-12 CLA; 11% cis-10,cis-12 CLA) was obtained from Nu-Chek Prep Inc. (Elysian, MN). The individual trans-10,cis-12 CLA and cis-9,trans-11 CLA isomers were purchased from Matreya, Inc. (Pleasant Gap, PA). Dulbecco’s modified Eagle’s medium, nutrient mixture F-12, dexamethasone, dihydroxyacetone phosphate (DHAP), isobutyl-methylxanthine, reduced NADH, gentamicin sulfate, HEPES buffer, hydrocortisone, insulin, and transferrin were purchased from Sigma Chemical (St. Louis, MO). Collagenase (type I) was purchased from Worthington Biochemical (Freehold, NJ), fetal bovine serum (FBS) from Intergen (Purchase, NY), and fungizone from Gibco BRL (Division of Life Technologies, Gaithersburg, MD).

Animals and Primary Culture
Two-day-old crossbred pigs (York x Landrace) were obtained from a commercial producer and killed by CO₂ asphyxiation in a manner approved by the Animal Care and Use Committee at Oregon State University. Stromal-vascular (S-V) cells were harvested by a collagenase digestion procedure as previously described (Suryawan and Hu, 1997). Aliquots of S-V cells were counted using a hemacytometer and seeded in culture dishes at a density of 5 x 10⁴ cells/cm² and incubated at 37°C in 5% CO₂ in air (designated d –1). Plating medium consisted of DME/F12 (1:1, vol/vol) containing 15 mmol/L of NaHCO₃, 15 mmol/L of HEPES buffer, and 50 mg/L of gentamicin sulfate supplemented with 10% FBS. Cells were plated on 96-well, six-well, or 10-cm plates to facilitate proliferation, differentiation, or gene expression studies, respectively. After 24 h, attached cells were washed three times with plating medium to remove unattached cells and cellular debris (designated d 0). After washing, cells were continually induced to differentiate in medium containing 10% FBS, 580 ng/ mL of insulin, 10 µg/mL of transferrin, 500 ng/mL of hydrocortisone, and the indicated fatty acid treatments until d 10. Fatty acids were prepared as indicated by Brodie et al. (1999) and were added to serum-containing differentiation medium before treating cells. The final concentration of dimethyl sulfoxide was less than 0.1% (vol/vol). Dimethyl sulfoxide alone did not affect markers of proliferation or differentiation at the levels present in these experiments. Serum was not analyzed for fatty acid content. Culture media were changed every 2 d until d 10 (except where stated otherwise), when cells were subjected to glycerol-3-phosphate dehydrogenase (GPDH) assays, oil red O staining, or gene expression analysis. By d 10 in differentiation medium, greater than 70% of the cells accumulated multilocular lipid droplets. Trypan blue exclusion tests were conducted to confirm cell viability, and no fatty acid treatment was associated with decreased viability at the concentrations used in these experiments.

Experimental Design
Stromal-vascular cells were plated as described above. To determine the effect of CLA isomers on the proliferation of porcine preadipocytes, cultures were continuously treated from d 0 to d 2 with 0 to100 µM of a CLA-mix, cis-9,trans-11 CLA, trans-10,cis-12 CLA, or linoleic acid in plating medium. Cell number was determined following 48 h of treatment (before confluence) based on the formation of formazan after 4 h of incubation with the tetrazolium salt WST-1. To determine the effect of CLA isomers on enzymatic and morphological markers of adipogenesis, as well as the mRNA transcript expression of adipocyte-related genes, cultures were continuously treated from d 0 to d 10 with 0 to 100 µM of a CLA-mix, cis-9,trans-11 CLA, trans-10,cis-12 CLA, or linoleic acid in differentiation medium. On d 10, treated cultures were either used for isolation of cell lysates to facilitate GPDH activity studies, isolation of mRNA to facilitate gene expression studies, or were stained with oil red O (ORO) to measure total lipid accumulation. For each experiment, individual fatty acid treatments were compared with equivalent vehicle-treated cultures which served as controls. Linoleic acid was used to control for potential effects of the addition of PUFA. Three to six replicates were performed for the described experiments, with cells harvested from a different pig for each replicate.

Oil Red O Staining
To qualitatively assess S-V cell differentiation by microscopy, cells were exposed to differentiation media from d 0 to 10 and then fixed in 10% formalin and stained with 0.3% ORO for lipid. Extractable ORO was then measured spectrophotometrically (570 nm) by modifying the procedure of Suryawan and Hu (1993). Briefly, the wells were fixed with Baker’s formalin for 15 min, rinsed with distilled water, equilibrated in 100% propylene glycol for 2 min, and then stained with ORO for 10 min. Wells were then treated with 60% propylene glycol (vol/vol) for 1 min to remove free ORO, and rinsed with distilled water. The ORO was extracted with the addition of isopropanol and ORO determined in aliquots from wells following shaking the culture plates 30 min at room temperature.

Glycerol-3-Phosphate Dehydrogenase Activity
The Sn-glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) activity was determined by measuring spectrophotometrically the disappearance of NADH during the GPDH-catalyzed reduction of DHAP under zero-order conditions by the method of Kozak and Jensen (1974), as modified by Wise and Green (1979). Briefly, differentiated cells were harvested in ice-cold lysate buffer (0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, 5 mM Tris base, pH 7.4). Membranes were disrupted by sonication and supernatant fractions were collected following centrifugation at 13,000 x g for 10 min at 4°C to remove cellular debris. The reaction was initiated by the addition of supernatant fractions to a standard mixture containing 100 mM triethanolamine/HCl buffer (pH 7.4), 2.5 mM EDTA, 0.176 mM NADH, 0.37 mM DHAP, and 0.1 mM ß-mercaptoethanol. The reaction was linear for sample time and concentration. Glycerol-3-phosphate dehydrogenase activity was expressed as units per milligram of protein, where one unit of activity is defined as the oxidation of 1 nmol of NADH/ min. Protein was measured according to Bradford (1976).

Cell Number Assay
The colorimetric assay for quantification of cell number and cell viability, based on the cleavage of the tetrazolium salt WST-1 (4-[3-{4-iodophenyl}-2-{4-nitrophenyl}-2H-5-tetrazolio]-1,3-benzene disulfonate) by mitochondrial dehydrogenases, was performed according to the manufacturer (catalog No. 1644 807; Boehringer Mannheim, Indianapolis, IN) as detailed by Brodie et al. (1999). The WST-1 assay was validated for the primary S-V cell system by verifying that increased S-V cell plating density correlated with increased formazan formation.

RNA Isolation
Cells were harvested with a cell scraper, and total RNA was extracted using the guanidinium-phenol-chloroform method (Chomczynski and Sacchi, 1987). Total RNA concentration was determined spectrophotometrically at 260 and 280 nm. The ratio of light absorbance at 260 nm to that at 280 nm was between 1.7 and 2.1 for all samples. Five micrograms of total RNA from each sample was separated on a 1.2% denaturing formaldehyde gel and stained with ethidium bromide. The RNA integrity was assessed visually by judging the quality of 18 and 28S rRNA bands.

Semiquantitative Reverse Transcription-PCR
Reverse transcription (RT) reaction solution (20 µL) consisted of 4 µg of total RNA, 50 U of SuperScript II reverse transcriptase (Invitrogen/Life Technologies, Carlsbad, CA), 40 U of an RNAse inhibitor (Invitrogen/ Life Technologies), 0.5 mmol/L of deoxyribonucleotide triphosphate, and 100 ng of random hexamer primers. Polymerase chain reaction was performed in 50 µL containing 20 mmol/L Tris·HCl, pH 8.4, 50 mmol/L KCl, 1.0 µL of RT reaction, 2.5 U of Platinum Taq DNA polymerase (Hot Start; Invitrogen/Life Technologies), 0.2 mmol/L of deoxyribonucleotide triphosphate, 2 mmol/L Mg²⁺ (Invitrogen/Life Technologies), 10 pmol each of gene specific primers, and 10 pmol each of primers specific for either ß-actin or 36B4. Thermal cycling parameters were as follows: one cycle at 94°C for 4 min, followed by 26 to 30 cycles at 94°C for 1 min, 56°C for 2 min, and 72°C for 2 min, with a final extension at 72°C for 8 min. Primers were synthesized at the Center for Gene Research at Oregon State University. Identity of PCR products was verified either by restriction digest analysis or DNA sequencing. The cycle number for each multiplex PCR reaction was selected by experimentally determining the highest cycle number in which the amplification of both cDNA products was within a linear range. The optimal cycle number was then considered to be two cycles lower than the highest cycle of linearity. The RT-PCR amplicons were visualized by separating DNA on a 3% agarose gel and staining with SYBR Green according to the manufacturer’s directions (Molecular BioProbes, Eugene, OR), followed by detection and quantification using the Kodak Digital Science (Rochester, NY) electrophoresis documentation and analysis system 120. Primer sequences, amplicon size and cycle length are listed in Table 1. Data for each replicate represented the mean of three individual RT-PCR.

	Results

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Proliferation
To study the potential isomer-specific effect of CLA on the proliferation of porcine S-V cells, the cleavage of the tetrazolium salt, WST-1, by mitochondrial dehydrogenases was measured on d 2 following treatment with 0 to 100 µmol/L of either CLA-mix, cis-9,trans-11 CLA, trans-10,cis-12 CLA, or linoleic acid. Proliferation was decreased in a dose-dependent fashion by trans-10,cis-12 CLA (P < 0.05). The CLA-mix, cis-9,trans-11 CLA, and linoleic acid did not affect cell number (Figure 1).

View larger version (19K): [in this window] [in a new window]   Figure 1. The effect of increasing doses of fatty acids on formazol formation in primary cultures of porcine preadipocytes following treatment for 2 d. Stromal-vascular cells were isolated from porcine adipose tissue, seeded at a concentration of 5 x 104 cells/cm2 in plating medium, and incubated for 24 h at 37°C (designated d –1). Cultures were then continuously treated with 0 to 100 µM of crude mixture of conjugated linoleic acid isomers (CLA-mix), trans-10,cis-12 CLA (10, 12-CLA), cis-9,trans-11 CLA (9,11-CLA), or linoleic acid (C18:2) in plating medium from d 0 to d 2. After 48 h of treatment, cell number was determined based on the formation of formazan after 4 h incubation with the tetrazolium salt, WST-1, as described in the Materials and Methods section. Data are means ± SEM from four experiments, each performed with cells harvested from a different pig. Six replicate wells were assayed within treatment for each pig. Means within a treatment that do not share a common asterisk differ, P < 0.05. Treatment effect was verified by counting cells from similarly treated cultures that were not exposed to WST-1.

View larger version (20K): [in this window] [in a new window]   Figure 2. The effect of increasing doses of fatty acids on glycerol-3-phosphate dehydrogenase (GPDH) activity in primary cultures of differentiating porcine preadipocytes on d 10. Stromal-vascular cells were isolated from porcine adipose tissue, seeded at a concentration of 5 x 104 cells/cm2 in plating medium, and incubated for 24 h at 37°C (designated d –1). Cultures were then continuously treated from d 0 to d 10 with 0 to 100 µM crude mixture of conjugated linoleic acid isomers (CLA-mix), the cis-9,trans-11 isomer of CLA (9, 11-CLA), the trans-10,cis-12 isomer of CLA (10, 12-CLA), or linoleic acid (C18:2) in differentiation medium. Cell lysates were harvested after 10 d of treatment and immediately assayed for GPDH activity. Data are means ± SEM from six experiments, each performed with cells harvested from a different pig. Three replicate wells were assayed within treatment for each pig. Means within a treatment that do not share a common asterisk differ, P < 0.05.

View larger version (42K): [in this window] [in a new window]   Figure 3. The effect of fatty acids on the number of lipid-filled adipocytes and accumulation of Oil Red O-stained material (OROSM) present in primary cultures of differentiating porcine preadipocytes on d 10 after induction of differentiation. Panel A: Stromal-vascular cells were isolated from porcine adipose tissue, seeded at a concentration of 5 x 104 cells/cm2 in plating medium, and incubated for 24 h at 37°C (designated d –1). Differentiating preadipocytes were continuously treated from d 0 to d 10 with vehicle, 100 µM crude mixture of conjugated linoleic acid isomers (CLA-mix), 25 µM trans-10,cis-12 CLA (10,12-CLA), 100 µM cis-9,trans-11 CLA (9,11-CLA), or 100 µM linoleic acid (C18:2). The microscopic magnification was 10x. Panel B: Stromal-vascular cells were isolated and treated as above. On d 10, plates were stained with Oil Red O, and the extracted stain was quantified spectrophotometrically. The amount of OROSM per well was expressed relative to the protein content of unstained wells receiving similar treatment on the same plate. Data are means ± SEM from three experiments, each performed with cells harvested from a different pig.

View larger version (14K): [in this window] [in a new window]   Figure 4. The effect of trans-10,cis-12 conjugated linoleic acid (10,12-CLA) on glycerol-3-phosphate dehydrogenase (GPDH) activity in the presence of increasing doses of cis-9,trans-11 CLA (9,11-CLA) in primary cultures of differentiating porcine preadipocytes on d 10. Stromal-vascular cells were isolated from porcine adipose tissue, seeded at a concentration of 5 x 104 cells/cm2 in plating medium, and incubated for 24 h at 37°C (designated d–1). Cultures were then continuously treated from d 0 to 10 with 12.5 µM 10, 12-CLA in the presence of 0 to 100 µM 9, 11-CLA from d 0 to d 10. Cell lysates were harvested on 10 d and immediately assayed for GPDH activity. Data are means ± SEM from three experiments, each performed with cells harvested from a different pig. Means that do not have a common letter differ, P < 0.05.

View larger version (53K): [in this window] [in a new window]   Figure 5. Isomer-specific effect of fatty acids on the expression of peroxisome proliferator-activated receptor (PPAR; A), CCAAT/enhancer binding protein (C/EBP; B), sterol regulatory element-binding protein-1c (SREBP-1C; C), and chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF; D) mRNA on d 10 in primary cultures of differentiating porcine preadipocytes. Stromal-vascular cells were isolated from porcine adipose tissue, seeded at a concentration of 5 x 104 cells/cm2 in plating medium, and incubated for 24 h at 37°C (designated d –1). Cultures were then continuously treated with vehicle, 100 µM crude mixture of conjugated linoleic acid isomers (CLA-mix), 100 µM cis-9,trans-11 CLA (9,11-CLA), 25 µM trans-10,cis-12 CLA (10,12-CLA), or 100 µM linoleic acid (C18:2) in differentiation medium from d 0 to 10. Total RNA was isolated and mRNA expression was measured using semi-quantitative reverse-transcription PCR as described in the Materials and Methods section. Data are means ± SEM from three experiments, each performed with cells harvested from a different pig. The PPAR and SREBP-1c values were normalized to ß-actin expression and C/EBP and COUP-TF values were normalized to 36B4 transcript expression. Means that do not share a common asterisk differ, P < 0.05. Amplicons from representative gels are depicted in the insets for each gene.

	Discussion

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Several studies indicate that feeding CLA to growing pigs decreases carcass fat, although the effect of CLA has been variable and occurs by an unknown mechanism (Azain et al., 2003; Dugan et al., 2004). The present study provides evidence that CLA inhibits fat cell differentiation in the pig, as suggested by the ability of CLA to potently inhibit several markers of differentiation in primary cultures of pig preadipocytes. This inhibitory action is isomer-specific as trans-10,cis-12 CLA potently prevented triglyceride accumulation and GPDH activity, whereas cis-9,trans-11 CLA did not affect these measurements. Inhibition of lipid accumulation and GPDH activity were correlated with the down-regulation of PPAR and SREBP-1c gene expression and the upregulation of COUP-TF1 gene expression.

In the current model of adipogenesis derived primarily from the study of clonal preadipocyte cell lines, the sequential expression of C/EBPß, PPAR, and C/EBP results in transactivation of adipocyte-specific genes leading to terminal differentiation of the preadipocyte and the induction of metabolic pathways related to lipid metabolism (Lazar, 2002). Presently, PPAR is considered the master regulator of adipocyte differentiation, whereas C/EBP is thought to potentiate differentiation by upregulating genes that confer insulin sensitivity on the adipocyte (Hamm et al., 1999; Lazar, 2002; Rosen et al., 2002). Additionally, SREBP-1c is believed to potentiate adipogenesis both by upregulating PPAR expression and by increasing availability of ligands for PPAR through upregulation of genes involved in lipid metabolism (Kim et al., 1998; Fajas et al., 1999). This developmental pattern of expression for adipogenic transcription factors is obscured in primary cultures of pig preadipocytes, as these cells express significant quantities of mRNA and protein for C/EBP , C/EBP ß, and PPAR before differentiation is initiated (Lee et al., 1998; Ding et al., 1999; Hausman 2000). This suggests that primary cultures of pig preadipocytes may be at a later stage of differentiation than clonal preadipocyte cell lines. Nonetheless, cross talk between C/EBP and PPAR seems to be necessary for porcine adipogenesis (Hausman, 2003). More research is needed to better understand how each of these proteins may regulate fat cell differentiation in the pig.

The inhibition of several markers of differentiation by CLA in the present study contrasts the findings of two earlier studies that also examined the effect of CLA in differentiating cultures of primary porcine preadipocytes. In the first study, cis-9,trans-11 CLA increased triglyceride accumulation compared with linoleic acid following 24 h of treatment, suggesting that CLA may stimulate adipogenesis in the pig (Ding et al., 2000). In that study, neither cis-9,trans-11 CLA nor trans-10,cis-12 CLA affected PPAR or CEBP gene expression. In the second study, CLA failed to affect triglyceride accumulation in serum-containing primary cultures, and there were no isomer-specific effects of CLA on PPAR mRNA abundance (McNeel and Mersmann, 2003). However, in that study, the ability to detect an inhibitory effect of CLA on differentiation may have been confounded by a low differentiation response in control cells. In the present study, preadipocyte differentiation was evaluated by measuring morphological data in conjunction with GPDH activity and the expression of marker genes. The acute effect of CLA on the differentiation of pig preadipocytes was not examined; however, treating differentiating cultures of porcine S-V cells with trans-10,cis-12 CLA for 10 d resulted in a consistent and dose-dependent inhibition for all indices examined. Conversely, cis-9,trans-11 CLA had no effect on GPDH activity or the expression of PPAR and C/ EBP mRNA abundance, suggesting that cis-9,trans-11 CLA did not affect the differentiation of pig preadipocytes. It is important to note that small differences in culture conditions may dramatically affect outcomes so conflicting results are occasionally reported by laboratories using similar in vitro approaches (Novakofski, 2004). The disparities between these two laboratories could be due to differences in genetics and gender, culture conditions (e.g., serum-free vs. serum systems, bovine serum albumin supplementation, fatty acid handling), duration of treatment, or source and lot number of culture components (e.g., serum, BSA, plastics). Interestingly, both gender and genetics have been shown to influence the effect that feeding CLA to pigs has on body composition (Azain, 2003). Data from the present study support the hypothesis that CLA inhibits the differentiation of pig preadipocytes in primary culture.

Trans-10,cis-12 CLA is now accepted as the active isomer responsible for body composition changes in rodents in vivo and for the antiadipogenic action of CLA in clonal preadipocyte cell lines and primary cultures of human preadipocytes (Brown et al., 2001, 2003; Kang et al., 2003). Because growth trials have predominantly used crude CLA preparations that contained a mixture of at least four cis/trans isomers, it is unclear which isomer underlies the observed decreases in body fat when CLA is fed to growing pigs. Our data support a role for trans-10,cis-12 CLA as an isomer that inhibits the proliferation and differentiation of pig preadipocytes and suggest that feeding the pure trans-10,cis-12 isomer may decrease body fat in pigs. It is clear from rodent studies that CLA isomers can have very distinct biological activities (Park et al., 1999; Pariza et al., 2001). Interestingly, some in vitro data have suggested that the cis-9,trans-11 CLA isomer might increase the differentiation of pig preadipocytes (Ding et al., 2000). Thus, the possibility exists that the cis-9,trans-11 CLA isomer may antagonize the action of the trans-10,cis-12 CLA in pig preadipocytes. Although a stimulatory action of cis-9,trans-11 CLA was not observed in the current study, presence of cis-9, trans-11 CLA tended to blunt the ability of trans-10,cis-12 CLA to inhibit GPDH activity. This finding suggests that the presence of cis-9,trans-11 CLA may decrease the potency of crude preparations of CLA. As a whole, these data suggest that feeding individual isomers of CLA may have merit.

In the present study, both the CLA-mix and trans-10,cis-12 CLA decreased PPAR mRNA abundance. These results agree with several studies in which CLA inhibited differentiation and decreased the mRNA abundance of PPAR in 3T3-L1 preadipocytes (Brodie et al., 1999; Evans et al., 2001, Kang et al., 2003). Brown et al. (2003) reported that CLA-induced inhibition of human preadipocyte differentiation also was accompanied by decreased PPAR mRNA abundance. Finally, feeding trans-10,cis-12 CLA to mice has consistently decreased PPAR mRNA (Kang and Pariza, 2001; Takahashi et al., 2002). Thus, there is a general consensus that CLA decreases the expression of PPAR mRNA abundance when inhibiting adipogenesis. Data from our study is consistent with this conclusion.

Unexpectedly, CLA inhibited adipogenesis independent of effects on C/EBP mRNA abundance in the present study; however, given the emerging role of PPAR as the master regulator of adipogenesis (Farmer et al., 2002; Rosen et al., 2002), effects on C/EBP gene expression may not be necessary to significantly inhibit fat cell differentiation. This idea is supported by recent work, where retinoids also were shown to inhibit the differentiation of pig preadipocytes independent of an effect on C/EBP mRNA abundance (Brandebourg and Hu, 2005). Furthermore, in clonal preadipocytes, SREBP-1c has been shown to regulate adipogenesis independently of effects on C/EBP through induction of PPAR expression and activity and through direct regulation of lipogenic genes (Kim and Spiegelman, 1996; Kim et al., 1998; Rosen et al., 2000). In the current study, SREBP-1c mRNA was decreased in an isomer-specific pattern that mirrored the effect of trans-10,cis-12 CLA on GPDH activity and paralleled the expression of PPAR. The down-regulation of SREBP-1c by trans-10,cis-12 CLA is consistent with the current model of adipogenesis. These data support a role for SREBP-1c in the mechanism by which CLA inhibits the differentiation of pig preadipocytes.

Chicken ovalbumin upstream promoter transcription factor is an orphan nuclear receptor that has recently been implicated as a potential negative regulator of adipogenesis in the pig (Brandebourg and Hu, 2005). In the present study, both the CLA-mix and trans-10,cis-12 CLA increased the expression of COUP-TF mRNA concomitant with the down-regulation of markers of preadipocyte differentiation, providing correlative evidence indicating that COUP-TF may play a role in the antiadipogenic action of CLA in pig preadipocytes. It is known that COUP-TF can compete with PPAR for both dimerization with RXR receptors and for binding to the putative PPAR direct repeat site in the promoter regions of target genes (Tsai and Tsai, 1997). Because binding of PPAR to response elements in the promoters of target genes represents a critical point in the regulation of gene transcription by PPAR, competition for DNA binding sites could be expected to significantly decrease the transcriptional activity of PPAR. Thus, although we did not examine the effect of CLA on the transcriptional activity of PPAR in the present study, a role for COUP-TF in the mechanism by which CLA inhibited the differentiation of porcine preadipocytes is consistent with the current model of adipogenesis.

	Implications

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Feeding conjugated linoleic acid mixtures to growing pigs decreases carcass fat, although this effect has been variable, and it occurs by an unknown mechanism. These data suggest that conjugated linoleic acid can inhibit the differentiation of pig preadipocytes in an isomer-specific manner, and this inhibition is correlated with the downregulation of peroxisome proliferator-activated receptor and sterol regulatory element-binding protein-1c messenger RNA. This study is the first to identify chicken ovalbumin upstream promoter-transcription factor 1 as a novel potential regulator of conjugated linoleic acid action. Understanding the isomer-specific action of conjugated linoleic acid on the adipocyte will help us to devise designer conjugated linoleic acid mixtures that, when fed to growing pigs, will result in higher quality pork products that are healthier to consume.

	Footnotes

 
¹ The authors thank Drahn Acres Farms for their assistance in support of this work and A. Menino for technical discussions.

² Correspondence: Univ. of Hawaii at Manoa, 3050 Maile Way, Gilmore Hall 202, Honolulu 96822-2279 (phone: 808-956-8131; fax: 808-956-9105; e-mail: hucy{at}ctahr.hawaii.edu).

Received for publication February 7, 2005. Accepted for publication June 2, 2005.

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