J. Anim Sci.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brandebourg, T. D.
Right arrow Articles by Hu, C. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brandebourg, T. D.
Right arrow Articles by Hu, C. Y.
J. Anim. Sci. 2005. 83:98-107
© 2005 American Society of Animal Science

ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

Regulation of differentiating pig preadipocytes by retinoic acid

T. D. Brandebourg* and C. Y. Hu{dagger},1

* Department of Animal Sciences and and {dagger} College of Agricultural Sciences, Oregon State University, Corvallis 97331


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
All-trans retinoic acid (ATRA) potently inhibits the differentiation of porcine preadipocytes in primary culture; however, the mechanism by which ATRA exerts this effect in pigs is poorly understood. The objective of this study was to use retinoid receptor-specific ligands to investigate the mechanism underlying the antiadipogenic action of retinoids in cultured pig preadipocytes by identifying the retinoid receptor mediating this action and examining the effect of retinoids on the expression of key adipogenic transcription factors. Stromal-vascular cells were harvested from porcine adipose tissue and cultured in serum-free medium. Glycerol-3-phoshphate dehydrogenase (GPDH) activity, a late marker of preadipocyte differentiation, was decreased (P < 0.01) by the addition of 0 to 10 µM of either ATRA, a nonspecific agonist for both the retinoic acid receptor (RAR) and the retinoid X receptor (RXR) or the selective RAR agonist, 4-(E-2-[5,6,7,8-tet-rahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid (TTNPB). Addition of increasing amounts of Ro-61, a RAR-specific antagonist (0 to 10 µM) prevented ATRA and TTNBP from decreasing GPDH activity. Addition of methoprene acid, an RXR-specific agonist, increased (P < 0.01) GPDH activity. Preadipocytes were then continuously treated with 10 nM of TTNPB in the presence or absence of 1 µM Ro-61, and mRNA was isolated on d 2 and 8. Addition of TTNPB decreased (P < 0.001) the expression of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), sterol regulatory element-binding protein-1c (SREBP-1c), retinoid X receptor {alpha} (RXR{alpha}), and adipocyte fatty acid binding protein (aP2) mRNA transcripts, whereas these effects were prevented by the presence of Ro-61. Interestingly, TTNBP increased (P < 0.001) the mRNA abundance of the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1), whereas Ro-61 prevented this increase. These changes were independent of alterations in the mRNA abundances of the retinoic acid receptor {alpha}, and CCAAT/enhancer binding protein {alpha} and ß (C/EBPß; C/EBP{alpha}) genes. These results indicate that retinoic acid inhibits porcine preadipocyte differentiation by a mechanism that involves activation of the RAR and downregulation of PPAR{gamma}, RXR{alpha}, and SREBP-1C mRNA. This mechanism is independent of changes in C/EBPß and C/EBP{alpha} mRNA abundance and may involve COUP-TF.

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


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Provitamin A carotenoids and all-trans retinoic acid (ATRA), an active metabolite of vitamin A, potently inhibit the differentiation of clonal preadipocyte cell lines (Chawla and Lazar, 1994Go; Xue et al., 1996Go; Kawada et al., 2000Go). Suryawan and Hu (1997)Go reported that ATRA also effectively inhibited the differentiation of porcine preadipocytes in primary culture, suggesting that retinoids may regulate fat cell differentiation in growing animals. This view is supported by the correlation between increased marbling and low serum retinol concentrations in Japanese Wagyu beef cattle fed vitamin A-deficient diets (Nakai et al., 1992Go; Torii et al., 1996Go).

The mechanism by which ATRA inhibits the differentiation of pig preadipocytes is unclear. The biological activity of retinoids is manifested through binding to intracellular retinoic acid receptors (RAR) or retinoid X receptors (RXR). Ligand-activated RAR-RXR or RXR-RXR dimers then regulate the expression of ATRA target genes (Mangelsdorf and Evans, 1995Go). Studies using 3T3-L1 preadipocytes suggest that ATRA inhibits adipogenesis in the mouse by downregulating the expression of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) and CCAAT/enhancer binding protein {alpha} (C/EBP{alpha}; Xue et al., 1994; Kawada et al., 2000Go). The effect of retinoids on the expression of these transcription factors in pig preadipocytes has not been explored.

Better understanding of how retinoids inhibit the differentiation of pig preadipocytes may provide insight into the regulation of adipose tissue development in vivo. Therefore, the objective of this study was to use retinoid receptor-specific compounds to investigate the underlying mechanism by which retinoids inhibit the differentiation of porcine preadipocytes in primary culture by identifying the retinoid receptor mediating this action and examining the effect of retinoids on the expression of key adipogenic transcription factors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
Materials
Dulbecco’s modified Eagle’s medium, nutrient mixture F-12, dihydroxyacetone phosphate (DHAP), reduced nicotinamide adenine dinucleotide (NADH), gentamicin sulfate, HEPES buffer, hydrocortisone, insulin, transferrin, and 4-(E-2-[5,6,7,8-tetrahydro-5,5,8,8-tet-ramethyl-2-naphthalenyl]-1-propenyl) benzoic acid (TTNPB), were purchased from Sigma Chemical Co. (St. Louis, MO). All-trans-retinoic acid, 9-cis-retinoic acid, and methoprene acid were purchased from Biomol (Plymouth Meeting, PA). Collagenase (type I) was purchased from Worthington Biochemical (Freehold, NJ), fetal calf serum from Intergen (Purchase, NY), and fungizone from Gibco BRL, a division of Life Technologies (Gaithersburg, MD). The RAR antagonist, Ro-61, was a kind gift from Roche Pharmaceuticals (Basal, Switzerland).

Animals and Cell Culture
Two-day-old crossbred pigs were obtained from a commercial producer (Drahn Acre Farms, Corvallis, OR) 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, 1997Go). Aliquots of S-V cells were counted by hemacytometry, seeded in either 35- or 100-mm culture dishes at a density of 5 x 104 cells/cm2, and incubated at 37°C in 5% CO2 in air. Plating medium consisted of 1,2-dimethoxyethane/F-12 (vol/vol; 1:1) containing 15 mM NaHCO3, 15 mM HEPES buffer, and 50 mg/L of gentamicin sulfate supplemented with 10% fetal calf serum. After 24 h, attached cells were washed three times using plating medium to remove unattached cells and cellular debris. After washing, cells were maintained in serum-free DME/F-12 medium containing 100 nM insulin, 10 µg/ mL of transferrin, and 50 ng/mL of hydrocortisone. Culture media was changed every 3 d until d 8, except where stated otherwise. Cells were subjected to GPDH assays on d 8, and subsets of cells were harvested on d 2 and 8 for oil red O (ORO) staining and gene expression analysis. By d 8 in culture medium, more than 70% of the cells had accumulated multilocular lipid droplets.

Experiment 1: Concentration-Dependent Effect of Receptor-Specific Retinoids on Glycerol-3-Phosphate Dehydrogenase (GPDH) Activity in Differentiating Porcine Preadipocytes
Stromal-vascular cells were isolated from porcine adipose tissue and then seeded in plating medium for 24 h at 37°C at a concentration of 5 x 104 cells/cm2 (designated d –1). Cultures were continuously treated from d 0 to 10 with 0 to 10 µM ATRA (a nonspecific agonist for both the RAR and the RXR), 9-cis-retinoic acid, methoprene acid (RXR-selective agonist), or TTNPB (RAR-selective agonist) in differentiation medium. Cell lysates were harvested after 10 d of treatment and immediately assayed for GPDH activity. Six replicates were performed each using cells harvested from a different pig. Three replicate wells were assayed within treatment for each pig.

Experiment 2: Concentration-Dependent Effect of the RAR Antagonist, Ro61, on the Ability of ATRA or TTNPB to Inhibit GPDH Activity in Primary Cultures of Differentiating Porcine Preadipocytes
Stromal-vascular cells were isolated from porcine adipose tissue and then seeded in plating medium for 24 h at 37°C at a concentration of 5 x 104 cells/cm2 (designated d –1). Cultures were continuously treated from d 0 to 10 with 0 to 10 µmol/L Ro61 in the presence of either 1 µM ATRA or 0.1 nM TTNPB. Cell lysates were harvested after 10 d of treatment and immediately assayed for GPDH activity. Six replicates were performed each using cells harvested from a different pig. Three replicate wells were assayed within treatment for each pig.

Experiment 3: The Effect of RAR-Selective Retinoids on the mRNA Transcript Expression of Adipocyte-Related Genes
Stromal-vascular cells were isolated from porcine adipose tissue and then seeded in plating medium for 24 h at 37°C at a concentration of 5 x 104 cells/cm2 (designated d –1). Cultures were continuously treated with vehicle, 0.1 nM TTNPB (RAR-selective agonist), or 0.1 nM TTNPB plus 1 µM/L Ro61 (RAR-selective antagonist). Total RNA was isolated from duplicate treated plates on either d 2 or 8 of culture. The mRNA abundance for the adipocyte fatty acid binding protein (aP2), PPAR{gamma}, sterol regulatory element-binding protein-1c (SREBP-1C), chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF), RXR{alpha}, RAR{alpha}, and the C/EBP{alpha} and C/EBPß genes was measured using semiquantitative reverse-transcription PCR (RT-PCR). Three replicates were performed each using cells harvested from a different pig. Three replicate PCR reactions were performed within treatment for each pig.

Histochemistry
In order to qualitatively assess S-V cell differentiation by microscopy, cells were exposed to induction media from d 0 to 8, and then fixed in 10% formalin, stained with ORO for lipid, and extractable ORO was measured spectrophotometrically (570 nm) by modifying the procedure of Suryawan and Hu (1993)Go. 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 for 1 min to remove free ORO and rinsed with distilled water. The ORO was extracted with 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-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)Go, as modified by Wise and Green (1979)Go. Briefly, differentiated cells were harvested in ice-cold lysate buffer (0.25 M sucrose, 1 mM EDTA, 1 mM dithiotheitol, 5 mM Tris base, pH 7.4). Membranes were disrupted by sonication and supernatants were collected following centrifugation at 13,000 x g for 10 min to remove cellular debris. The reaction was initiated by the addition of supernatants 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 mg of protein where one unit of activity is defined as the oxidation of 1 nmol NADH/min. Protein was measured according to Bradford (1976)Go.

RNA Isolation
Cells were harvested with a cell scraper and total RNA was extracted using the guanidinium-phenol-chloroform method (Chomczynski and Sacchi, 1987Go). Total RNA concentration was determined spectrophotometrically using A260 and A280 measurements. 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 RT-PCR
Reverse transcription 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 deoxynucleotide triphosphate, and 100 ng of random hexamer primers. Polymerase chain reaction was performed in 50 µL containing 20 mmol/L of Tris•HCL, pH 8.4, 50 mmol/L of KCl, 1.0 µL of reverse transcription reaction, 2.5 U of Platinum Taq DNA polymerase (Hot Start, Invitrogen/ Life Technologies), 0.2 mmol/L of deoxynucleotide triphosphate, 2 mmol/L of 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 94°C for 4 min, followed by 26 to 30 cycles, 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 via DNA sequencing. 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 Electrophoresis Documentation and Analysis System 120 (Rochester, NY). Primer sequences, amplicon size, and cycle length are listed in Table 1Go. Data for each replicate represented the mean of three individual RT-PCR.


View this table:
[in this window]
[in a new window]
  		Table 1. Oligonucleotide polymerase chain reaction primers
 
Statistical Analyses
Data are expressed as the mean ± SEM. Each replicate consisted of a single batch of S-V cells harvested from the subcutaneous adipose tissue of an individual pig. Data were analyzed by using one-way ANOVA followed by multiple comparisons of means with Fisher’s LSD using SAS (SAS Inst., Inc., Cary, NC). Gene transcription data was analyzed using two-way ANOVA, with day and treatment as main effects. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
Differentiation
Glycerol-3-phosphate dehydrogenase activity was used as a marker of differentiation because GPDH activity is expressed in terminally differentiated, mature fat cells, but it is not present in undifferentiated S-V cells. Continually treating S-V cells with increasing amounts of either ATRA or 9c-RA (10 nM to 10 µM) decreased GPDH activity in a concentration-dependent fashion (P < 0.01; Figure 1Go). To determine which retinoid receptor mediated this effect, receptor-specific retinoids were tested for their affect on GPDH activity. The RAR-specific retinoid agonist, TTNPB, (10 pM to 10 µM) potently decreased GPDH activity with as little as 100 pM (P < 0.001). The addition of the RXR-specific agonist, methoprene acid (10 nM to 10 µM) increased GPDH activity (P < 0.01; Figure 1Go). Addition of Ro-61 (10 pM to 10 µM) reversed the ability of both ATRA and TTNBP to decrease GPDH activity (Figure 2Go).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 1. The effect of increasing doses of retinoids 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 with 0 to 10 µM all-trans retinoic acid (ATRA), 9-cis retinoic acid (9cRA), methoprene acid (MA; retinoid X receptor-selective agonist), or 4-(E-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid (TTNPB; retinoic acid receptor-selective agonist) 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.

 


View larger version (16K):
[in this window]
[in a new window]
  		Figure 2. The effect of the retinoic acid receptor antagonist, Ro61, on the ability of all-trans retinoic acid (ATRA) or 4-(E-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid (TTNPB) to inhibit glycerol-3-phoshphate 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 with 0 to 10 µmol/L Ro61 in the presence of either: A) 1 µM ATRA, or B) 0.1 nM TTNPB from d 0 to 10. Data are means ± SEM from six experiments, each performed with cells harvested from a different pig. Means that do not have a common letter differ, P < 0.05.

 
Differentiation was also measured by staining cells with ORO and then measuring the quantity of extractable stain. The quantity of ORO-stained material (OR-OSM) increased in cultures from d 0 to 8. In agreement with the GPDH activity data, as little as 10 nM TTNPB dramatically decreased the quantity of OROSM present in cultures of differentiating pig preadipocytes vs. the control (Figure 3Go), and the addition of Ro-61 reversed this effect. Methoprene acid numerically increased the amount of OROSM present in cultures of differentiating pig preadipocytes relative to the control.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. The effect of retinoids on the accumulation of oil red O-stained material (ORSM) 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 either vehicle, 10 µM methoprene acid (MA), 10 nM 4-(E-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid (TTNPB), or 10 nM TTNPB + 10 µM Ro61. At the indicated times, plates were stained with oil red O, and the extracted stain was quantified spectrophotometrically. The amount of ORSM 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.

 
Gene Expression
Total mRNA was extracted from cells following either 2 or 8 d of treatment by either dimethyl sulfoxide (carrier), 10 nM TTNPB, or 10nM TTNPB plus 1 µM Ro-61. The RAR-selective agonist TTNPB decreased (P < 0.008) the mRNA abundance for the aP2 gene relative to the control on both d 2 and 8 (Figure 4AGo), with the effect being greater (P < 0.001) on d 8. The addition of Ro-61 reversed this effect. Similarly, TTNPB decreased (P <0.001) the mRNA abundance for the PPAR{gamma} gene relative to the control on both d 2 and 8 (Figure 4BGo), and this effect was reversed by the addition of Ro-61 treatment. Likewise, TTNPB decreased (P < 0.001) the mRNA abundance for SREBP-1c relative to the control on both d 2 and 8 (Figure 4CGo), and the addition of Ro-61 reversed this effect. Interestingly, TTNPB increased (P < 0.001) the mRNA abundance for the COUP-TF1 gene on both d 2 and 8 (Figure 4DGo). Addition of Ro-61 reversed this effect. Meanwhile, TTNPB decreased (P < 0.001) the mRNA abundance for RXR{alpha} relative to the control on both d 2 and 8 (Table 2Go), whereas the addition of Ro-61 reversed this effect. However, TTNPB had no effect on the mRNA abundances for the RAR{alpha}, C/EBP{alpha}, and C/EBPß genes on any day measured (Table 2Go). Likewise, the addition of Ro-61 had no effect on the mRNA for these genes. There were no significant interactions between day and treatment for any gene measured.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 4. The effect of retinoids on the expression of adipocyte fatty acid binding protein (aP2, panel A), peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}, panel B), sterol regulatory element-binding protein-1c (SREBP-1C, panel C), and chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF, panel D) mRNA on d 2 and 8 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, 0.1 nM 4-(E-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid (TTNPB), or 0.1 nM TTNPB plus 1 µM/L Ro61 from d 0 to d 8. Total RNA was isolated on the indicated days, and mRNA expression was measured using semiquantitative reverse-transcription PCR. Data are means ± SEM from three experiments, each performed with cells harvested from a different pig. Means with an asterisk differ (P < 0.05) from other means within the same day. Amplicons from representative gels are depicted in the insets for each gene.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Effect of retinoids on the steady-state mRNA abundance of retinoid receptors and CCAAT/enhancer binding protein {alpha} and ß genes on d 2 and 8 in primary cultures of differentiating porcine preadipocytes
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
Suryawan and Hu (1997)Go determined that ATRA potently inhibited the differentiation of pig preadipocytes though the underlying mechanism was not elucidated. The present study indicates that retinoic acid must activate the RAR receptor to decrease several markers of differentiation in primary cultures of pig preadipocytes (GPDH activity, aP2 gene expression and lipid accumulation). This inhibitory activity of retinoids was correlated with decreases in PPAR{gamma}, RXR{alpha}, and SREBP-1c mRNA, but it was independent of changes in CAAT-enhancer binding protein mRNA steady-state concentrations. Furthermore, an upregulation of COUP-TF1 mRNA abundance was correlated with decreases in markers of adipogenesis, suggesting that COUP-TF1 may play a role in the antiadipogenic action of retinoids.

Retinoids regulate cellular functions by binding to intracellular RAR or RXR. Retinoic acid selectively binds to RAR leading to the formation of RAR-RXR heterodimer. Alternatively, 9-cis-retinoic acid, which can be generated by intracellular isomerization of ATRA (Levin et al., 1992Go; Heyman et al., 1992Go), can bind to either RAR or RXR with relatively high affinity, leading to the formation of a RAR-RXR heterodimer or a RXR-RXR homodimer. These receptor dimers then bind cis-acting DNA elements to regulate target gene expression (Leid et al., 1992Go; Mangelsdorf et al, 1994Go; Mangelsdorf and Evans, 1995Go). The expression of the RAR{alpha} gene in pig adipocytes was confirmed for the first time as mRNA transcripts for RAR{alpha} were detected in primary cultures of porcine S-V cells derived from adipose tissue throughout the culture period. In agreement with Ding et al. (1999)Go, RXR{alpha} mRNA transcripts were detected at the initiation of differentiation and their steady-state concentration remained high until the cultures were terminated on d 10 after induction.

Because treatment with ATRA could potentially lead to the activation of both RAR and RXR in pig preadipocytes, our first objective was to use RAR- or RXR-selective retinoids to determine which receptor family mediated the antiadipogenic activity of ATRA. The RAR-specific retinoid, TTNPB, was 1,000-fold more effective at inhibiting adipocyte differentiation than either ATRA or 9-cis-retinoic acid, as determined using GPDH activity as a marker of differentiation (Figure 1Go). Furthermore, the antiadipogenic activity of TTNPB was blocked by addition of Ro-61 (Figure 2Go). In contrast to RAR agonists, the RXR-specific retinoid, methoprene acid, stimulated the differentiation of pig preadipocytes (Figure 1Go). We conclude the antiadipogenic action of retinoids is mediated by the RAR receptor system. This conclusion is supported by a previous study, where it was determined that RAR receptors mediated the anti-adipogenic effect of ATRA in mouse-derived 3T3-L1 pre-adipocytes (Xue et al., 1996Go).

Our second objective was to investigate the effect of retinoid administration on the expression of transcription factors that are known to regulate adipogenesis. In the current model derived primarily from the study of clonal preadipocyte cell lines, the sequential induction of C/EBPß, PPAR{gamma}, and C/EBP{alpha} in preadipocytes results in transactivation of adipocyte-specific genes leading to terminal differentiation of the adipocyte (Lazar, 2002Go). During this process, ligand-activated PPAR{gamma} forms a heterodimer with RXR{alpha}, resulting in the induction of PPAR{gamma}-responsive genes. However, it is now clear that significant differences in the expression of adipogenic transcription factors exist between clonal cell lines and primary cultures of pig preadipocytes. Primary porcine preadipocytes express significant quantities of mRNA and protein for C/EBP{alpha}, -ß, and -{delta}, as well as PPAR{gamma}, before differentiation is initiated, which obscures the developmental pattern of their expression (Lee et al., 1998Go; Ding et al., 1999Go; Hausman, 2000Go). However, although PPAR{gamma} protein expression does not precede the expression of C/EBP{alpha} in primary cultures of porcine preadipocytes, in agreement with clonal cell line models, the cross talk between these two proteins seems to be necessary for porcine adipogenesis (Hausman, 2003Go).

In the present study, TTNPB decreased the expression of PPAR{gamma}, SREBP-1c, RXR{alpha}, and aP2 mRNA transcripts in differentiating pig preadipocytes. These results are consistent with one study where the treatment of primary cultures of porcine S-V cells with ATRA for 3 d resulted in inhibition of PPAR{gamma} protein (Kim et al., 2000Go). However, TTNPB-induced changes in the present study were independent of alterations in the mRNA abundances of the RAR{alpha} and C/EBP{alpha} genes. Work using mouse-derived 3T3-L1 preadipocytes as a model indicates that ATRA inhibits adipogenesis in clonal cells by down regulating PPAR{gamma} and C/EBP{alpha} protein, while not preventing the initial induction of C/ EBPß (Schwarz et al., 1997Go). These changes seem to be the result of the down regulation of gene expression as subsequent studies have indicated that ATRA treatment decreases the mRNA abundance of the PPAR{gamma}, C/EBP{alpha}, RXR{alpha}, and RAR{alpha} but not C/EBPß genes in 3T3-L1 preadipocytes (C. Y. Hu, unpublished data; Kawada et al., 2000Go). These divergent effects of retinoids on RAR{alpha} and C/EBP{alpha} mRNA expression may be due in part to differences in the developmental stage of the preadipocyte between our primary system and clonal cell lines, heterogeneity of cell types in primary cultures of adipose tissue-derived S-V cells vs. homogeneity of cell lines, the timing and duration of serum exposure, or species-specific differences. These differences highlight the limitations of extrapolating between clonal cell lines and species of interest.

Interestingly, the mRNA expression of adipogenic transcription factors was rather stable between d 2 and 8 of culture in the present study. This contrasts with previous reports, in which the mRNA abundances of adipocyte-related genes such as lipoprotein lipase, fatty acid synthase, and glucose transporter 4 increased between 5- and 30-fold over a 10-d period following the induction of differentiation of porcine S-V cells in primary cultures (Ding et al., 1999Go; McNeel et al., 2000Go). However, in those studies, the increased mRNA abundances for adipogenic transcription factors were less robust than increases observed for functional genes. For instance, Ding et al. (1999)Go reported there was no change in expression of SREBP-1C, RXR{alpha}, or C/EBP{alpha} on d 7 compared with d 0 of culture. McNeel et al. (2000)Go reported that C/EBPß mRNA expression was unchanged following 7 d in culture; however, C/EBP{alpha} mRNA significantly increased compared with d 0 in that study. Ding et al. (1999)Go reported a onefold induction of PPAR{gamma} mRNA during 10 d of culture, although differences between d 2 and 7 were significantly smaller. In agreement with those studies, C/EBPß, SREBP-1C, and RXR{alpha} mRNA were unchanged between d 2 and 8 of culture in the present study. However, PPAR{gamma} mRNA abundance was not altered by day, and C/EBP{alpha} and aP2 mRNA transcripts numerically increased only modestly (13 and 16%, respectively) between d 2 and 8 of culture. Although these studies share similar culture protocols, several factors may have contributed to the observed differences in gene expression. For instance, age and genetics of the pigs used by the two laboratories differed. Sex was not reported in these studies, so uncharacterized sex effects may have been present as well. Furthermore, plating density is known to affect the homeostasis of preadipocyte cultures, and differences in the plating density existed between studies (Novakofski, 1987Go). Finally, subtle differences may arise from differences in medium components (e.g., serum, dimethyl sulfoxide carrier), plastics, and culture environment. These differences notwithstanding, generally the results in the current study agree with data reported previously, although in the present study, the expression of PPAR{gamma}, C/EBP{alpha}, and aP2 mRNA transcripts was affected only modestly by day in culture.

The downregulation of PPAR{gamma} and SREBP-1c in response to retinoids observed in the present study is consistent with the current model of adipogenesis. Based on research using clonal cell lines, PPAR{gamma} is now considered the master regulator of adipocyte differentiation, whereas C/EBP{alpha} is thought to potentiate differentiation by up-regulating genes that confer insulin sensitivity to the adipocyte (Hamm et al., 1999Go; Lazar, 2002Go; Rosen et al., 2002Go). Currently, SREBP-1c is believed to potentiate adipogenesis both by upregulating PPAR{gamma} expression and by increasing the availability of ligands for PPAR{gamma} by upregulating genes involved in lipid metabolism (Kim et al., 1998Go; Fajas et al., 1999Go). Thus, it is conceivable that adipogenesis could be effectively inhibited through a mechanism that is independent of effects on the expression of either C/EBPß or C/EBP{alpha}, especially if the mechanism targets PPAR{gamma}. Clearly C/EBP{alpha} expression is essential for adipogenesis in the pig (Yu and Hausman, 1998Go; Hausman, 2000Go). Although C/EBP{alpha} mRNA expression remains relatively constant as adipogenesis progresses, PPAR{gamma} mRNA expression increases throughout culture concomitant with differentiation (Ding et al., 1999Go; McNeel et al., 2000Go; present study). Given that differentiation of pig preadipocytes was dramatically inhibited in the absence of significant changes in expression of C/EBP{alpha}, it is tempting to speculate that C/EBP{alpha} may play a permissive role in regulating the final stages of terminal differentiation of pig preadipocytes, whereas PPAR{gamma} and SREBP-1c may play more significant regulatory roles. Currently, the mechanism underlying crosstalk between C/EBP{alpha} and PPAR{gamma} is poorly characterized during pig preadipocyte differentiation, and more research is needed to better understand the regulatory roles of these two proteins.

Chicken ovalbumin upstream promoter transcription factor is an orphan nuclear receptor that has been implicated in mediating the ATRA inhibition of differentiation in multiple cell types (Widom et al., 1992Go; Jonk et al., 1994Go; Van der Wees et al., 1996Go). In the present study, TTNPB increased the expression of COUP-TF mRNA concomitant with the inhibition of markers of preadipocyte differentiation, providing correlative evidence suggesting that COUP-TF1 may play a role in the antiadipogenic action of retinoids in pig preadipocytes. It is known that COUP-TF can compete with PPAR{gamma} for both dimerization with RXR receptors and for binding to the same direct repeat site in the promoter regions of target genes (Tsai and Tsai, 1997Go). Because PPAR{gamma} must bind to RXR to be transcriptionally active, even slight changes in the kinetics of RXR binding would be expected to significantly affect the transcriptional activity of PPAR{gamma}. Furthermore, initiation is the rate-limiting step in transcription. Thus, DNA binding of PPAR{gamma} to response elements in target genes represents a critical point in the regulation of gene transcription by PPAR{gamma}, and competition for DNA binding sites could be expected to significantly decrease the transcriptional activity of PPAR{gamma}. In this regard, Brodie et al. (1996)Go observed that ATRA both induced the expression of COUP-TF and increased the binding of COUP-TF to a PPAR{gamma} binding sequence transfected into 3T3-L1 preadipocytes. In the present study, TTNPB increased the expression of COUP-TF mRNA, and this effect was blocked by the addition of the RAR receptor antagonist, Ro-61, in a manner that was parallel to the ability of retinoids to inhibit the differentiation of pig preadipocytes. Thus, although we did not examine the effect of retinoids on the transcriptional activity of PPAR{gamma} in the present study, a role for COUP-TF in the mechanism by which retinoids inhibited the differentiation of porcine preadipocytes is consistent with the current model of adipogenesis. This work provides a rationale for further studying the potential of COUP-TF as a regulator of porcine adipogenesis.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
This is the first study that shows the inhibitory effect of retinoic acid on pig preadipocyte differentiation in primary culture is mediated by the retinoic acid receptor {alpha} and is correlated with the down regulation of peroxisome proliferator-activated receptor {gamma}, retinoid X receptor {alpha}, and sterol regulatory element-binding protein-1c mRNA. Furthermore, this study is the first to identify chicken ovalbumin upstream promoter-transcription factor 1 as a novel potential regulator of fat cell differentiation in the pig. Understanding the action of retinoic acid will help us to devise an effective method to control adipose tissue development in meat-producing animals, so that meat products can be provided that are healthier to consume and more cost-effective to produce.

1 Correspondence: 138 Strand Ag Hall (phone: 541-737-1915; fax: 541-737-4574; e-mail: ChingYuan.Hu{at}orst.edu).

Received for publication June 21, 2004. Accepted for publication October 12, 2004.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72:248–254.[Medline]

Brodie, A. E., V. A. Manning, and C. Y. Hu. 1996. Inhibitors of preadipocyte differentiation induce COUP-TF binding to a PPAR/RXR binding sequence. Biochem. Biophys. Res. Commun. 228:655–661.[Medline]

Brodie, A. E., V. A. Manning, K. R. Ferguson, D. E. Jewell, and C. Y. Hu. 1999. Conjugated linoleic acid inhibits differentiation of pre- and post-confluent 3T3-L1 preadipocytes but inhibits cell proliferation only in preconfluent cells. J. Nutr. 129:602–606.[Abstract/Free Full Text]

Chawla, A., and M. A. Lazar. 1994. Peroxisome proliferator and retinoid signaling pathways co-regulate preadipocyte phenotype and survival. Proc. Natl. Acad. Sci. USA 91:1786–1790.[Abstract/Free Full Text]

Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid-guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156–159.[Medline]

Ding, S. T., R. L. McNeel, and H. J. Mersmann. 1999. Expression of porcine adipocyte transcripts: Tissue distribution and differentiation in vitro and in vivo. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 123:307–318.[Medline]

Fajas L., K. Schoonjans, L. Gelman, J. B. Kim, J. Najib, G. Martin, J.-C. Fruchart, M. Briggs, B. M. Spiegelman, and J. Auwerx. 1999. Regulation of peroxisome proliferator-activated receptor gamma expression by adipocyte differentiation and determination factor 1/sterol regulatory element binding protein 1: Implications for adipocyte differentiation and metabolism. Mol. Cell. Biol. 19:5495–5503.[Abstract/Free Full Text]

Hamm, J. K., A. K. el Jack, P. F. Pilch, and S. R. Farmer. 1999. Role of PPAR gamma in regulating adipocyte differentiation and insulin-responsive glucose uptake. Ann. NY Acad. Sci. 892:134–145.[Abstract/Free Full Text]

Hausman, G. J. 2000. The influence of dexamethasone and insulin on expression of CCAAT/enhancer binding protein isoforms during preadipocyte differentiation in porcine stromal-vascular cell cultures: Evidence for very early expression of C/EBP{alpha}. J. Anim. Sci. 78:1227–1235.[Abstract/Free Full Text]

Hausman, G. J. 2003. Dexamethasone induced preadipocyte recruitment and expression of CCAAT/enhancing binding protein {alpha} and peroxisome proliferator activated receptor-{gamma} proteins in porcine stromal-vascular (S-V) cell cultures obtained before and after the onset of fetal adipogenesis. Gen. Comp. Endocrinol. 133:61–70.[Medline]

Heyman, R. A, D. J. Mangelsdorf, J. A. Dyck, R. B. Stein, G. Eichele, R. M. Evans, and C. Thaller. 1992.9-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell 68:397–406.[Medline]

Jonk L. J., M. E. de Jonge, C. E. Pals, S. Wissink, J. M. Vervaart, J. Schoorlemmer, and W. Kruijer. 1994. Cloning and expression during development of three murine members of the COUP family of nuclear orphan receptors. Mech. Dev. 47:81–97.[Medline]

Kawada, T., Y. Kamei, A. Fujita, Y. Hida, N. Takahashi, E. Sugimoto, and T. Fushiki. 2000. Carotenoids and retinoids as suppressors on adipocyte differentiation via nuclear receptors. Biofactors 13:103–109.[Medline]

Kim, H. S., G. J. Hausman, D. B. Hausman, R. J. Martin, and R. G. Dean. 2000. The expression of peroxisome proliferator-activated receptor gamma in pig fetal tissue and primary stromal-vascular cultures. Obes. Res. 8:83–88.[Medline]

Kim, J. B., H. M. Wright, M. Wright, and B. M. Spiegelman. 1998. ADD1/SREBP1 activates PPAR gamma through the production of endogenous ligand. Proc. Natl. Acad. Sci. USA 95:4333–4337.[Abstract/Free Full Text]

Kozak, L. P., and J. T. Jensen. 1974. Genetic and developmental control of multiple forms of l-glycerol 3-phosphate dehydrogenase. J. Biol. Chem. 249:7775–7781.[Abstract/Free Full Text]

Lazar, M. A. 1999. PPAR{gamma} in adipocyte differentiation. J. Anim. Sci. 77(Suppl 3):16–22.

Lazar, M. A. 2002. Becoming fat. Genes Dev. 16:1–5.[Free Full Text]

Lee, K., G. J. Hausman, and R. G. Dean. 1998. Expression of C/ EBP alpha, beta and delta in fetal and postnatal subcutaneous adipose tissue. Mol. Cell. Biochem. 178:269–274.[Medline]

Leid, M., P. Kastner, R. Lyons, H. Nakshatri, M. Saunders, T. Zacharewski, J.Y. Chen, A. Staub, J. M. Garnier, and S. Mader. 1992. Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently. Cell 68:377–395.[Medline]

Levin, A.A., L. J. Sturzenbecker, S. Kazmer, T. Bosakowski, C. Huselton, G. Allenby, J. Speck, C. Kratzeisen, M. Rosenberger, and A. Lovey. 1992.9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha. Nature 355:359–361.[Medline]

Mangelsdorf, D. J., and R. M. Evans. 1995. The RXR heterodimers and orphan receptors. Cell 83:841–850.[Medline]

Mangelsdorf, D. J., K. Umesono, and R. M. Evans. 1994. The retinoid receptors. Pages 319–349 in The Retinoids: Biology, Chemistry, and Medicine. M. B. Sporn, A. B. Roberts, and D. S. Goodman, ed. Raven Press Ltd., New York.

McNeel, R. L., S. T. Ding, E. O. Smith, and H. J. Mersmann. 2000. Expression of porcine adipocyte transcripts during differentiation in vitro and in vivo. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 126:291–302.[Medline]

Nakai, A., K. Hasegawa, and M. Hiramitsu. 1992. Vitamin a deficiency in Japanese black cattle. J. Clin. Vet. Med. 10:2143–2148.

Novakofski, J., and C.Y. Hu. 1987. Culture of isolated adipose tissue cells. J. Anim. Sci. 65(Suppl. 2):12–19.

Rosen, E. D., C.-H. Hsu, X. Wang, S. Sakai, M. W. Freeman, F.J. Gonzalez, and B. M. Spiegelman. 2002. C/EBP-{alpha} induces adipogenesis through PPAR{gamma}: A unified pathway. Genes Dev. 16:22–26.[Abstract/Free Full Text]

Schwarz, E. J., M. J. Reginato, D. Shao, S. L. Krakow, and M. A. Lazar. 1997. Retinoic acid blocks adipogenesis by inhibiting C/ EBP-beta-mediated transcription. Mol. Cell. Biol. 17:1552–1561.[Abstract]

Suryawan, A., and C. Y. Hu. 1993. Effect of serum on differentiation of porcine adipose stromal-vascular cells in primary culture. Comp. Biochem. Physiol. Comp. Physiol. 105:485–492.[Medline]

Suryawan, A., and C. Y. Hu. 1997. Effect of retinoic acid on differentiation of cultured pig preadipocytes. J. Anim. Sci 75:112–117.[Abstract/Free Full Text]

Torii, S., T. Matsui, and H. Yano. 1996. Development of intramuscular fat in Wagyu beef cattle depends on adipogenic or antiadipogenic substances present in serum. Anim. Sci. 63:73–78.

Tsai, S. Y., and M. J. Tsai. 1997. Chick ovalbumin upstream promoter-transcription factors (COUP-TFS): Coming of age. Endocrinol. Rev. 18:229–240.[Abstract/Free Full Text]

van der Wees, J., P. J. Matharu, K. de Roos, O. H. Destree, S. F. Godsave, A. J. Durston, and G. E. Sweeney. 1996. Developmental expression and differential regulation by retinoic acid of Xenopus COUP-TF-A and COUP-TF-B. Mech. Dev. 54:173–184.[Medline]

Widom, R. L., M. Rhee, and S. K. Karathanasis. 1992. Repression by ARP-1 sensitizes apolipoprotein AI gene responsiveness to RXR alpha and retinoic acid. Mol. Cell. Biol. 12:3380–3389.[Abstract/Free Full Text]

Wise, L. S., and H. Green. 1979. Participation of one isozyme of cytosolic glycerophosphate dehydrogenase in the adipose conversion of 3T3 cells. J. Biol. Chem. 254:273–275.[Abstract/Free Full Text]

Xue, J. C., E. J. Schwarz, A. Chawla, and M. A. Lazar. 1996. Distinct stages in adipogenesis revealed by retinoid inhibition of differentiation after induction of PPARgamma. Mol. Cell. Biol. 16:1567–1575.[Abstract]

Yu, Z. K., and G. J. Hausman. 1998. Expression of CCAAT/enhancer binding proteins during porcine preadipocyte differentiation. Exp. Cell. Res. 245:343–349.[Medline]


This article has been cited by other articles:


Home page
 J. Lipid Res.Home page
M. Camacho, C. Rodriguez, J. Salazar, J. Martinez-Gonzalez, J. Ribalta, J.-R. Escudero, L. Masana, and L. Vila
Retinoic acid induces PGI synthase expression in human endothelial cells
J. Lipid Res., August 1, 2008; 49(8): 1707 - 1714.
[Abstract] [Full Text] [PDF]

Home page
 J ANIM SCIHome page
A. M. Arnett, M. E. Dikeman, C. W. Spaeth, B. J. Johnson, and B. Hildabrand
Effects of vitamin A supplementation in young lambs on performance, serum lipid, and longissimus muscle lipid composition
J Anim Sci, November 1, 2007; 85(11): 3062 - 3071.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
P. Gupta, S. W. Park, M. Farooqui, and L.-N. Wei
Orphan nuclear receptor TR2, a mediator of preadipocyte proliferation, is differentially regulated by RA through exchange of coactivator PCAF with corepressor RIP140 on a platform molecule GRIP1
Nucleic Acids Res., April 1, 2007; 35(7): 2269 - 2282.
[Abstract] [Full Text] [PDF]

Home page
 GeneticsHome page
W. Barendse, R. J. Bunch, J. W. Kijas, and M. B. Thomas
The Effect of Genetic Variation of the Retinoic Acid Receptor-Related Orphan Receptor C Gene on Fatness in Cattle
Genetics, February 1, 2007; 175(2): 843 - 853.
[Abstract] [Full Text] [PDF]

Home page
 J ANIM SCIHome page
T. D. Brandebourg and C. Y. Hu
Isomer-specific regulation of differentiating pig preadipocytes by conjugated linoleic acids
J Anim Sci, September 1, 2005; 83(9): 2096 - 2105.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brandebourg, T. D.
Right arrow Articles by Hu, C. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brandebourg, T. D.
Right arrow Articles by Hu, C. Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS