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© 2008 American Society for Nutrition J. Nutr. 138:455-461, March 2008

Biochemical, Molecular, and Genetic Mechanisms

Trans-10, Cis-12 Conjugated Linoleic Acid Antagonizes Ligand-Dependent PPAR{gamma} Activity in Primary Cultures of Human Adipocytes1,2

Arion Kennedy3, Soonkyu Chung4, Kathleen LaPoint3, Oluwatoyin Fabiyi3 and Michael K. McIntosh3,*

3 Department of Nutrition, University of North Carolina, Greensboro, NC 27402-6170 and 4 Department of Pathology, Wake Forest University, School of Medicine, Winston Salem, NC 27157

* To whom correspondence should be addressed. E-mail: mkmcinto{at}uncg.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
We previously demonstrated that trans-10, cis-12 (10,12) conjugated linoleic acid (CLA) causes human adipocyte delipidation, insulin resistance, and inflammation in part by attenuating PPAR{gamma} target gene expression. We hypothesized that CLA antagonizes the activity of PPAR{gamma} in an isomer-specific manner. 10,12 CLA, but not cis-9, trans-11 (9,11) CLA, suppressed ligand-stimulated activation of a peroxisome proliferator response element-luciferase reporter. This decreased activation of PPAR{gamma} by 10,12 CLA was accompanied by an increase in PPAR{gamma} and extracellular signal-related kinase (ERK)1/2 phosphorylation, followed by decreased PPAR{gamma} protein levels. To investigate if 10,12 CLA-mediated delipidation was preventable with a PPAR{gamma} ligand (BRL), cultures were treated for 1 wk with 10,12 CLA or 10,12 CLA + BRL and adipogenic gene and protein expression, glucose uptake, and triglyceride (TG) were measured. BRL cosupplementation completely prevented 10,12 CLA suppression of adipocyte fatty acid-binding protein, lipoprotein lipase, and perilipin mRNA levels without preventing reductions in PPAR{gamma} or insulin-dependent glucose transporter 4 (GLUT4) expression, glucose uptake, or TG. Lastly, we investigated the impact of CLA withdrawal in the absence or presence of BRL for 2 wk. CLA withdrawal did not rescue CLA-mediated reductions in adipogenic gene and protein expression. In contrast, BRL supplementation for 2 wk following CLA withdrawal rescued mRNA levels of PPAR{gamma} target genes. However, the levels of PPAR{gamma} and GLUT4 protein and TG were only partially rescued by BRL. Collectively, we demonstrate for the first time, to our knowledge, that 10,12 CLA antagonizes ligand-dependent PPAR{gamma} activity, possibly via PPAR{gamma} phosphorylation by ERK.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Dysfunction of adipose tissue can result in insulin resistance and lipodystrophy. One major regulator in the development and function of adipose tissue is PPAR{gamma}, which induces the expression of a host of adipogenic genes such as lipoprotein lipase (LPL), insulin-stimulated glucose transporter 4 (GLUT4),5 perilipin (PLIN), and adipocyte fatty acid-binding protein (aP2). Mutations of PPAR{gamma} in humans are associated with insulin resistance and lipodystrophy (1,2). PPAR{gamma} null cells exhibit impaired adipogenesis (3) and dominant negative mutations in PPAR{gamma} inhibit adipogenesis (4). Thus, PPAR{gamma} activity is essential in adipose tissue for glucose uptake and triglyceride (TG) accumulation.

Regulation of PPAR{gamma} occurs through a variety of proposed mechanisms, including covalent modification by phosphorylation, ligand binding, and heterodimerization with the retinoic acid receptor (RXR) (5,6). Phosphorylation of PPAR{gamma} by activation of the mitogen-activated protein kinase (MAPK) pathway has been reported to inhibit adipogenesis (7). It has been demonstrated that phosphorylation of Ser-112 of PPAR{gamma}2 results in its ubiquination and proteosome degradation (8). Activation of PPAR{gamma} by natural (i.e. PUFA) or synthetic ligands such as thiazolidinediones (TZD) initiates heterodimerization with RXR followed by their binding to peroxisome proliferator response element (PPRE) in the promoters of adipogenic target genes. The TZD are hypoglycemics that activate PPAR{gamma}, leading to upregulation of adipogenic genes, thereby enhancing insulin sensitivity. Natural ligands of PPAR{gamma}2 such as cis-PUFA or prostaglandins such as PGJ2 (9) have a relatively low affinity for PPAR{gamma} compared with TZD. In contrast, SFA and certain trans PUFA such as conjugated linoleic acid (CLA) have been reported to impair insulin sensitivity, possibly by decreasing the expression of PPAR{gamma} and many of its downstream target genes (10–13).

CLA consists of dienoic isomers of linoleic acid, including trans-10, cis-12 CLA and cis-9, trans-11 CLA. CLA decreases body fat mass in animals (14) and some humans (15). Our group has demonstrated that trans-10, cis-12 CLA decreases adipogenic gene expression and the TG content of human (pre)adipocytes (10,11). We have also demonstrated that activation of MAPK kinase (MEK)/extracellular signal-related kinase (ERK) (11) and nuclear factor {kappa}B (NF{kappa}B) (16) signaling by trans-10, cis-12 CLA were essential for its suppression of adipogenic gene expression and delipidation in human adipocytes. A number of side effects have been associated with trans-10, cis-12 CLA supplementation in humans, such as insulin resistance, hyperglycemia, and dyslipidemia (17,18). Dyslipidemia, insulin resistance, and hyperglycemia are similar characteristics found in humans with mutations in PPAR{gamma}. Two recent reports by Belury et al. (19,20) showed that the PPAR{gamma} agonist rosiglitazone prevents or attenuates inflammation, lipodystrophy, and insulin resistance in mice fed a crude mixture of CLA isomers containing equal amounts of cis-9, trans-11 CLA and trans-10, cis-12 CLA. However, the isomer-specific mechanism by which CLA suppresses the expression of PPAR{gamma} and its target genes in human adipocytes remains to be elucidated. To address this issue, we examined the impact of CLA on PPAR{gamma} in the absence and presence of the PPAR{gamma} ligand rosiglitazone (BRL).


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Materials. All cell culture ware were purchased from Fisher Scientific. Western Lightning chemiluminescence substrate was purchased from Perkin Elmer Life Science. The 1-step RT-PCR kit used in semiquantitative mRNA analysis was purchased from Qiagen. Immunoblotting buffers, precast gels, and gene-specific primers were purchased from Invitrogen and ribosomal 18S competimer technology internal standards and DNA-free were purchased from Ambion. Polyclonal GLUT4 antibody was a gift from Drs. S. Cushman and X. Chen (NIDDK, NIH, Bethesda, MD). aP2 antibody was a gift from Dr. D. Bernlohr (University of Minnesota). Monoclonal antibodies for PPAR{gamma} (sc7273) and polyclonal antibodies for anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc20357) and β-actin (sc1616) were obtained from Santa Cruz Biotechnology. Anti-phospho (Thr-202/204) and total ERK antibodies were purchased from Cell Signaling Technologies. Cy3- and fluorescein-conjugated immunoglobulin G were purchased from Jackson Immunoresearch. Fetal bovine serum was purchased from Cambrex/BioWhittaker. BRL was a gift from Glaxo Smith Kline. Isomers of CLA (+98% pure) were purchased from Matreya. The Nucleofector and Dual Glo luciferase (luc) kits were obtained from Amaxa and Promega, respectively. All other reagents and chemicals were purchased from Sigma Chemical unless otherwise stated.

    Culturing of human primary adipocytes. Abdominal white adipose tissue was obtained from nondiabetic females between the ages of 20 and 50 y old with a BMI ≤ 30 during abdominoplasty with consent from the Institutional Review Board at the University of North Carolina at Greensboro. Tissue was digested using collagenase and stromal vascular cells were isolated as previously described (11). Experimental treatment of cultures containing ~50% preadipocytes and ~50% adipocytes occurred on d 12 of differentiation. Each experiment was conducted in duplicate and repeated at least 3 times using a mixture of cells from 2–3 subjects unless otherwise indicated.

    Preparation of fatty acids. Both isomers of CLA were complexed to fatty acid-free (≥98%) bovine serum albumin (BSA) at a 4:1 molar ratio using 1 mmol/L BSA stocks.

    Immunoblotting. Immunoblotting was conducted as previously described (11). To resolve PPAR{gamma} phospho-proteins, total cell extracts (75 µg protein) were subjected to 10% SDS-PAGE (acrylamide:bisacrylamide, 100:1, wt:wt) containing 4 mol/L urea and to electrophoresis at 80 V for 20 h as we previously described (21). Separated proteins were subsequently transferred to polyvinylidene difluoride membranes and immunoblotted with a monoclonal PPAR{gamma} antibody. For determining the phosphorylation status of PPAR{gamma}, a portion of the cell extracts from BSA vehicle and CLA treatment were incubated with 20 U of calf intestinal phosphatase (Cip) for 30 min at 37°C and for 15 min at 55°C. Subsequently, the samples were subjected to SDS-PAGE containing urea as described above.

    Immunostaining of PPAR{gamma}. Cells were cultured on coverslips for immunofluorescence microscopy and stained as described previously (11) except for the permeabilization step. Fixed cells were permeabilized with 0.1% Triton X-100 for 1 min on ice. Monoclonal anti-PPAR{gamma} (1:10) were incubated overnight at 4°C. Fluorescent images were captured with a SPOT digital camera mounted on an Olympus BX60 fluorescence microscope.

    Transient transfections of human adipocytes. For measuring PPAR{gamma} activity, primary human adipocytes were transiently transfected with the multimerized PPAR-responsive (luc) reporter construct pTK-PPRE3x-luc (22) using the Amaxa Nucleofector as previously described (21). On d 6 of differentiation, 1 x 106 cells from a 60-mm plate were trypsinized and resuspended in 100 µL of nucleofector solution (Amaxa) and mixed with 2 µg of pTK-PPRE3x-luc and 25 ng pRL-CMV for each sample. Electroporation was performed using the V-33 nucleofector program (Amaxa). Cells were replated in 96-well plates after 10 min of recovery in calcium-free RPMI media. Firefly luc activity was measured using the Dual-Glo luc kit and normalized to Renilla luc activity from the cotransfected control pRL-CMV vector. All luc data are presented as a ratio of firefly luc to Renilla luc activity. We consistently obtained ~75% transfection efficiency revealed by parallel transfections with a green fluorescent protein reporter construct. Both adipocytes and nonadipocytes were transfectable using this protocol based on aP2 immunostaining and 4',6-diamidino-phenylindole nuclear staining.

    RNA analysis. Following treatment, cultures were harvested for total RNA using Tri-Reagent according to the manufacturer's protocol. Contaminating DNA was removed with DNAase (DNA-free, Ambion). One microgram of RNA from each sample was used for semiquantitative RT-PCR using the One-Step RT-PCR kit (Qiagen) as previously described in Brown et al. (10). The gene-specific primer pairs used were previously described (10).

    Lipid staining. Lipid staining of cultures of human adipocytes was conducted as previously described (10) using Oil Red O (ORO).

    [3H] 2-deoxy-glucose uptake. Newly differentiated cultures of adipocytes were incubated with BSA vehicle, 30 µmol/L cis-9, trans-11 CLA, 30 µmol/L trans-10, cis-12 CLA, 30 µmol/L trans-10, cis-12 CLA + 1 µmol/L BRL, or 1 µmol/L BRL in adipocyte media for 2 d. Then, for an additional 2 d, cultures were incubated in 1 mL of serum-free basal DMEM containing 1000 mg/L D-(+)-glucose with or without 20 pmol/L of human insulin with BSA vehicle, 30 µmol/L cis-9, trans-11 CLA, 30 µmol/L trans-10, cis-12 CLA, 30 µmol/L trans-10, cis-12 CLA + 1 µmol/L BRL, or 1 µmol/L BRL in adipocyte media for another 2 d. Following the experimental treatments, insulin-stimulated uptake of [3H]-2-deoxy-glucose was measured following a 90-min incubation in the presence of 100 nmol/L human insulin as described previously (16).

    Statistical analysis. Statistical analyses were performed for data in Figure 1 testing the main effects of BRL and CLA and the interaction of the 2 (BRL x CLA) using 2-way ANOVA (JMP version 6.03, SAS Institute). Analyses for significant differences for data in Figure 4C were conducted using 1-way ANOVA. Student's t tests were used to compute individual pairwise comparisons of least square means (P < 0.05). Data are expressed as the means ± SE.


Figure 1
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  		FIGURE 1  Trans-10, cis-12 CLA blocks ligand-induced activation of PPAR{gamma} in human adipocytes. Cultures of newly differentiated human adipocytes were transfected on d 6 with pTK-PPRE3x-luc and pRL-CMV. Twenty four hours later, transfected cells were treated with dimethyl sulfoxide vehicle control (C), 30 µmol/L trans-10, cis-12 CLA, or 30 µmol/L cis-9, trans-11 CLA in the absence or presence of 0.1 µmol/L BRL for 24 h. Values are means ± SEM, n = 3. Means without a common letter differ, P < 0.05.

 

Figure 4
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  		FIGURE 4  Trans-10, cis-12 CLA antagonizes ligand-activated PPAR{gamma} expression, glucose uptake, and TG accumulation in human adipocytes. (A) Cultures of newly differentiated human adipocytes were treated for 1 wk with either BSA vehicle (B), 30 umol/L cis-9, trans-11 CLA (9), 30 µmol/L trans-10, cis-12 CLA (10), or 30 µmol/L trans-10, cis-12 CLA + 1 µmol/L BRL (10*) and then harvested. RNA was isolated and the mRNA levels of PPAR{gamma}2, aP2, LPL, PLIN, and Glut 4 were measured using semiquantitative RT-PCR. 18S ribosomal RNA was used as an internal control. (B) Cultures were treated as in A for 1 wk and then cell extracts were isolated and immunoblotted for PPAR{gamma}, aP2, Glut4, and β-actin. (C) Cultures were treated as in A for 4 d and then insulin-stimulated uptake of [3H]-2-deoxy-glucose was measured. Values are means ± SEM; n = 6. Means without a common letter differ, P < 0.05. (D) Cultures were treated as in A for 1 wk and then stained with ORO and phase-contrast photomicrographs were taken using an Olympus inverted microscope with a 10x objective. Data in A, B, and D are representative of 2–3 independent experiments.

 

   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Trans-10, cis-12 CLA decreases the activity and increases phosphorylation of PPAR{gamma}. To determine the extent to which CLA decreased PPAR{gamma} activity, basal- and ligand-induced activation of PPAR{gamma} activity were examined. There were no significant differences in basal levels of PPAR{gamma} activity due to CLA treatment in the absence of BRL (Fig. 1). However, PPAR{gamma} activity in BRL-stimulated cultures (+BRL) was lower in cultures treated with 30 µmol/L trans-10, cis-12 CLA compared with control and 30 µmol/L cis-9, trans-11 CLA-treated cultures. The extent to which trans-10, cis-12 CLA decreased PPAR{gamma} activity (~40%) was comparable to that of PPAR{gamma} antagonist GW9662, which inhibited ligand-induced PPAR{gamma} activity without affecting basal activity (data not shown).

Given the inverse relationship between PPAR{gamma} activity and its phosphorylation status (7), we wanted to determine the kinetics of PPAR{gamma} phosphorylation during treatment with trans-10, cis-12 CLA. Trans-10, cis-12 CLA caused a band shift in PPAR{gamma}1/2 after 24 h of treatment (Fig. 2A). Intriguingly, robust ERK1/2 phosphorylation at 24 h accompanied the PPAR{gamma}1/2 band shift, consistent with ERK1/2's role as a donor of phosphate groups to nuclear PPAR{gamma}1/2 and with our published data demonstrating that ERK1/2 is required for CLA's suppression of adipogenic gene expression and glucose uptake (11). However, because a PPAR{gamma} band shift could be due to processes other than phosphorylation (e.g. by acetylation, methylation, or sumylation), Cip was added to the cell extracts to remove phosphorylated groups. Trans-10, cis-12 CLA-induced band shifts of PPAR{gamma}1/2 were either lowered or attenuated by phosphatase treatment (Fig. 2B). Taken together, these data suggest that trans-10, cis-12 CLA promotes PPAR{gamma} and ERK phosphorylation, which contributes at least in part to CLA's isomer-specific reduction of PPAR{gamma} activity.


Figure 2
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  		FIGURE 2  Trans-10, cis-CLA increases PPAR{gamma} phosphorylation in human adipocytes. (A) Cultures of newly differentiated human adipocytes were serum starved for 24 h and then treated without (0) or with 30 µmol/L trans-10, cis-12 CLA (10) for 2, 4, 8, or 24 h. Subsequently, cell extracts were harvested, proteins separated by SDS-PAGE urea, and immunoblotted for the phosphorylated and unphosphorylated forms of PPAR{gamma}1/2, ERK1/2, and GAPDH (load control). (B) Cultures were treated for 24 h with BSA vehicle or 30 µmol/L trans-10, cis-12 CLA (10). A portion of the cell extracts from BSA vehicle and CLA treatment were incubated with Cip. Proteins were separated with SDS-PAGE urea and probed with antibodies targeting PPAR{gamma}1/2 and GAPDH. Data in A and B are representative of 2 independent experiments.

 
    Trans-10, cis-12 CLA decreases the protein levels of PPAR{gamma}. We previously demonstrated that a physiological level (e.g. 30 µmol/L) of trans-10, cis-12 CLA decreased the mRNA levels of PPAR{gamma} and several of its target genes in differentiating cultures of human stromal vascular cells (10) and in the newly differentiated cultures of human adipocytes (11). However, the isomer-specific impact of CLA on PPAR{gamma} protein levels in human adipocytes is unknown. PPAR{gamma}2 protein levels were decreased after 4 d and undetectable after 6 d of treatment with trans-10, cis-12 CLA compared with the BSA vehicle or cis-9, trans-11 CLA-treated cultures (Fig. 3A). Consistent with these data, newly differentiated cultures treated with 30 µmol/L trans-10, cis-12 CLA for 4 d had dramatically less nuclear PPAR{gamma} staining compared with BSA vehicle-treated cultures (Fig. 3B). These data demonstrate that trans-10, cis-12 CLA decreases PPAR{gamma} protein levels in an isomer-specific manner in newly differentiated human adipocytes.


Figure 3
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  		FIGURE 3  Trans-10, cis-12 CLA decreases PPAR{gamma} protein levels in human adipocytes. (A) Cultures of newly differentiated human adipocytes were treated with BSA vehicle (B), 30 µmol/L cis-9, trans-11 CLA (9), or 30 µmol/L trans-10, cis-12 CLA (10) for 2, 4, 6, or 8 d. Cells extracts were immunoblotted for PPAR{gamma}. To identify PPAR{gamma}1/2 in cultures of human adipocytes, cell extracts from 3T3-L1 adipocytes (mouse) were isolated and immunoblotted for PPAR{gamma}. A 3rd band was identified in human adipocytes and labeled as nonspecific (NS). (B) Cultures were treated with BSA vehicle (B) or 30 µmol/L trans-10, cis-12 CLA (10) for 4 d. PPAR{gamma} was detected using immunofluorescence microscopy. Data are representative of 2 independent experiments.

 
    Chronic effects of a trans-10, cis-12 CLA in the presence of a PPAR{gamma} ligand. To further evaluate the antagonistic effects of trans-10, cis-12 CLA on PPAR{gamma} activity, we examined the extent to which cosupplementation with the PPAR{gamma} agonist BRL could prevent trans-10, cis-12 CLA suppression of adipogenic genes and proteins, glucose uptake, and TG accumulation. Trans-10, cis-12 CLA decreased the mRNA (Fig. 4A) and protein (Fig. 4B) levels of PPAR{gamma}, aP2, LPL, and GLUT4 compared with BSA vehicle controls or cis-9, trans-11 CLA. Although BRL cosupplementation prevented CLA-mediated reductions in aP2, LPL, and PLIN gene expression, it did not prevent CLA suppression of PPAR{gamma}2 or GLUT4 mRNA levels (Fig. 4A). Consistent with these data, BRL cosupplementation prevented CLA suppression of aP2 protein expression but did not prevent CLA suppression of PPAR{gamma} or GLUT4 protein levels (Fig. 4B).

CLA isomer-specific reduction of insulin-stimulated glucose uptake (Fig. 4C) or TG accumulation (Fig. 4D) was not prevented by cosupplementation with BRL. Collectively, these data demonstrate that trans-10, cis-12 CLA chronically suppresses adipogenic gene and protein expression, glucose uptake, and TG content, which are only partially prevented by a PPAR{gamma} ligand.

    Effects of withdrawal from trans-10, cis-12 CLA in presence of a PPAR{gamma} ligand. Next, we wanted to determine whether the delipidating effects of CLA could be rescued by CLA withdrawal in the absence or presence of a PPAR{gamma} ligand. Surprisingly, withdrawal of trans-10, cis-12 CLA treatment for 2 wk did not restore the mRNA levels of LPL, PLIN, or GLUT4 gene (group 1, Fig. 5A) or the protein levels of PPAR{gamma} or GLUT4 (group 1, Fig. 5B). Interestingly, the pattern of gene and protein expression in group 1 was almost identical to that of the cultures treated for 1 wk with trans-10, cis-12 CLA (Fig. 4), indicating the effects of CLA were sustained over 2 wk. Consistent with these gene and protein data, cultures treated with trans-10, cis-12 CLA had less stainable TG 2 wk after withdrawal (group 1, Fig. 5C) compared with controls.


Figure 5
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  		FIGURE 5  Effects of withdrawal of trans-10, cis-12 CLA in the presence of a PPAR{gamma} ligand in human adipocytes. Cultures of newly differentiated human adipocytes were treated for 1 wk with either BSA vehicle (B), 30 µmol/L cis-9, trans-11 CLA (9), 30 µmol/L trans-10, cis-12 CLA (10), or 30 µmol/L trans-10, cis-12 CLA + 1 umol/L BRL (10*) and then had their treatments withdrawn for 2 wk (group 1, -BRL), or were treated with 1 µmol/L BRL for 2 wk during CLA withdrawal (group 2, +BRL). (A) RNA was isolated and the mRNA levels of PPAR{gamma}2, aP2, LPL, PLIN, and Glut4 were measured using semiquantitative RT-PCR. 18S ribosomal RNA was used as an internal control. (B) Cell extracts were isolated and immunoblotted for PPAR{gamma}, aP2, Glut4, and β-actin. (C) Cultures were stained with ORO and phase-contrast photomicrographs were taken using an Olympus inverted microscope with a 10x objective. Data are representative of 2–3 independent experiments.

 
BRL supplementation for 2 wk following CLA withdrawal rescued PPAR{gamma}, aP2, and LPL gene expression compared with BSA vehicle- or cis-9, trans-11 CLA-treated cultures, whereas PLIN and GLUT4 were partially rescued (Group 2, Fig. 5A). BRL supplementation for 2 wk following CLA withdrawal reversed or attenuated trans-10, cis-12 CLA suppression of aP2 and GLUT4 protein levels, respectively, compared with cultures not receiving BRL for 2 wk (group 1). Although CLA-treated cultures supplemented for 2 wk with BRL (group 2) had more PPAR{gamma} protein compared with those not receiving BRL for 2 wk (group 1), PPAR{gamma} protein levels did not return to the levels of the BSA vehicle- or cis-9, trans-11 CLA-treated cultures. Similarly, supplementation of cultures with BRL for 2 wk during CLA withdrawal (group 2) increased the TG content of cultures treated with trans-10, cis-12 CLA compared with cultures not receiving BRL during withdrawal (group 1). Interestingly, BRL was only effective in preventing delipidation when it was cosupplemented with CLA and then supplemented for another 2 wk following CLA withdrawal (group 2, Fig. 5C). Taken together, these data demonstrate that trans-10, cis-12 CLA-mediated delipidation persists after CLA withdrawal and is relatively refractory to supplementation with a PPAR{gamma} ligand unless the ligand is supplemented during and after CLA treatment.


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
The PPAR{gamma} agonist rosiglitazone has been demonstrated to prevent or attenuate inflammation, lipodystrophy, and insulin resistance in mice fed a crude mixture of CLA isomers (e.g. primarily cis-9, trans-11 CLA and trans-10, cis-12 CLA) (19,20). These data suggest an antagonism between one or both CLA isomers and PPAR{gamma}. However, the isomer-specific mechanism by which CLA suppresses the activity of PPAR{gamma} in human adipocytes remains unknown. We demonstrate in this article that trans-10, cis-12, but not cis-9, trans-11, CLA attenuates ligand-induced activation of PPAR{gamma} (Fig. 1), possibly via phosphorylation of PPAR{gamma} by ERK1/2 (Fig. 2). Inactivation of PPAR{gamma} leads to suppression of protein and mRNA levels of PPAR{gamma} and several of its target genes in newly differentiated human adipocytes (Figs. 3–5). BRL cosupplementation did not prevent insulin resistance caused by trans-10, cis-12 CLA (Fig. 4). Furthermore, we show that trans-10, cis-12 CLA-mediated suppression of TG accumulation does not return to control levels following CLA withdrawal or by supplementation with a PPAR{gamma} agonist following CLA treatment (Fig. 5). Only BRL cosupplementation followed by 2 wk of BRL supplementation restored the TG content of trans-10, cis-12 CLA-treated cultures to control levels. Taken together, these data provide further support for the concept that CLA's antiadipogenic effects in humans are due to the trans-10, cis-12 isomer and not the cis-9, trans-11 isomer and are directly linked to the suppression of PPAR{gamma} activity, adipogenic gene and protein expression, insulin-stimulated glucose uptake, and TG content, which appears to be due in part to an antagonism of ligand-mediated activation of PPAR{gamma}.

Potential mechanisms explaining the isomer-specific attenuation of PPAR{gamma} activity by CLA are shown in our working model (Fig. 6). We propose that trans-10, cis-12 CLA, a metabolite, or a signal activated by CLA suppresses PPAR{gamma} activity by: 1) phosphorylating PPAR{gamma} via activation ERK1/2; 2) inhibiting ligand activation and heterodimer formation with RXR; or 3) impairing DNA binding of the PPRE to target genes, thereby decreasing adipogenic gene transcription, insulin-stimulated glucose uptake, and TG synthesis.


Figure 6
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  		FIGURE 6  Working model CLA, metabolites, or signals suppress PPAR{gamma} activity by: 1) phosphorylating PPAR{gamma} via activation of NF{kappa}B and ERK1/2; 2) inhibiting ligand activation and/or heterodimer formation with RXR; or 3) impairing transcriptional activation of target genes, thereby decreasing TG synthesis.

 
    PPAR{gamma} phosphorylation. Support for the first 2 mechanisms comes from our discovery that trans-10, cis-12 CLA suppresses adipogenic gene expression and metabolism through activation of ERK1/2 (11) and NF{kappa}B (16). Reports demonstrating that NF{kappa}B (23–27) and MAPK (28–30) activation hinders PPAR{gamma} DNA-binding affinity or transcriptional activation provides a potential mechanism by which trans-10, cis-12 CLA suppresses the expression of PPAR{gamma} target genes, leading to delipidation. ERK1/2 activates NF{kappa}B (31) and inactivates PPAR{gamma} (32), resulting in its ubiquination and proteosome degradation (33). Further support comes from studies showing that PPAR{gamma} agonists attenuate cytokine-mediated inflammation by suppressing NF{kappa}B and/or MAPK signaling (34–37). Clearly, the activity of PPAR{gamma} is regulated by its phosphorylation status via phosphatases and kinases (33). Indeed, the phosphorylation of PPAR{gamma} at a consensus MAPK site within its A/B domain (e.g. Ser-112) by ERK1/2 or c-Jun-NH2-terminal kinase reduces its transcriptional activation potential, leading to insulin resistance and/or decreased adipogenesis (8,28–30,32,37,38). Interestingly, we previously reported that the MEK/ERK inhibitor U0126 blocked trans-10, cis-12 CLA suppression of adipogenic genes and glucose and fatty acid uptake (11). Consistent with these data, we found that trans-10, cis-12 CLA simultaneously increased the phosphorylation of ERK1/2 and PPAR{gamma} (Fig. 2A). Based on these data, our working hypothesis is that trans-10, cis-12 CLA antagonizes PPAR{gamma}'s activity acutely and PPAR{gamma} expression chronically in adipocytes via NF{kappa}B and ERK1/2 activation, leading to decreased glucose and fatty acid uptake and TG synthesis.

    Ligand binding. CLA may also compete with endogenous (i.e. unsaturated fatty acids) or exogenous (i.e. rosiglitazone-BRL) ligands for activation of PPAR{gamma}. Low affinity PPAR{gamma} ligands such as PUFA increase PPAR{gamma} activity and target gene expression (39). Several CLA isomers, including cis-9, trans-11 CLA, have been shown to be ligands for PPAR{gamma} (13,40) or its partner RXR (39). Consistent with the reported antagonism between PPAR{gamma} and inflammation, cis-9, trans-11 CLA has been shown to suppress NF{kappa}B activation and inflammatory cytokine production by lipopolysaccharide in dendritic cells (41) and in white adipose tissue of obese mice (42). However, we found that cosupplementation of trans-10, cis-12 CLA-treated cultures with up to 30 µmol/L cis-9, trans-11 CLA did not reverse insulin resistance or adipogenic gene expression (data not shown). In contrast, Granlund et al. (13) demonstrated that both cis-9, trans-11 CLA and trans-10, cis-12 CLA decreased the activity of a darglitazone-stimulated, LXR{alpha}-PPRE-LUC reporter in a dose-dependent manner up to 25 µmol/L in COS-1 cells and 3T3-L1 cells. We have also demonstrated in 3T3-L1 that both CLA isomers antagonize ligand-induced activation of PPAR{gamma} (10). Alternatively, CLA phosphorylation of PPAR{gamma} in the A/B domain could reduce PPAR{gamma} affinity for ligand and/or cofactor recruitment (33).

    Transcriptional activation. Another possible mechanism by which CLA reduces PPAR{gamma} activity is by impairing DNA binding of the PPAR{gamma}/RXR heterodimer itself to the PPRE in target genes, thereby decreasing transcriptional activation. Conceptually, this would lead to decreased lipogenesis and TG accumulation. However, chromatin immunoprecipitation studies are needed to support this speculative mode of action of CLA.

One possible explanation for the long-term effects of CLA following withdrawal could be that CLA accumulates within the phospholipid and neutral lipid fractions of the cell, as we have previously shown for both isomers (10). Thus, CLA could continue to antagonize PPAR{gamma}/RXR activity following withdrawal, thereby impacting endogenous ligand production, phosphorylation, and/or directly interfering with their transcriptional activation.

In summary, although cosupplementation with BRL, a high affinity ligand for PPAR{gamma}, generally prevented or attenuated trans-10, cis-12 CLA suppression of adipogenic gene and protein expression and TG content, it did not prevent CLA's suppression of a PPAR{gamma} reporter construct or insulin-stimulated glucose uptake. Furthermore, BRL supplementation for 2 wk after CLA withdrawal did not completely rescue its antiadipogenic and TG-lowering effects. Taken together, these data suggest that trans-10, cis-12 CLA may decrease PPAR{gamma} activity acutely by increasing PPAR{gamma} phosphorylation via ERK1/2 and chronically by decreasing PPAR{gamma} transcription, thereby decreasing the amount PPAR{gamma} available for ligand binding, leading to the suppression of insulin-stimulated glucose uptake and TG accumulation.


   ACKNOWLEDGMENTS
 
We thank Dr. Maria Sandberg Boysen for guidance in conducting the urea gel for identifying PPAR{gamma} band shifts due to phosphorylation.


   FOOTNOTES
 
1 Supported by grants from the NIH (2R01DK 063070) and the North Carolina Agriculture Research Service (NCARS 06520) to M.K.M., and by the NIH (F31DK076208) to A.J.K. Back

2 Author disclosures: A. Kennedy, S. Chung, K. LaPoint, O. Fabiyi, and M. K. McIntosh, no conflicts of interest. Back

5 Abbreviations used: aP2, adipocyte-specific fatty acid-binding protein; BSA, bovine serum albumin; Cip, calf intestinal phosphatase; CLA, conjugated linoleic acid; ERK, extracellular signal-related kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUT4, insulin-dependent glucose transporter 4; HBSS, Hanks balanced salt solution; LPL, lipoprotein lipase; luc, luciferase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; NF{kappa}B, nuclear factor kappa B; ORO, Oil Red O; PLIN, perilipin; PPRE, peroxisome proliferator response element; RXR, retinoic acid receptor; TG, triglyceride; TZD, thiazolidinedione. Back

Manuscript received 14 September 2007. Initial review completed 3 November 2007. Revision accepted 11 December 2007.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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