J. Anim Sci. 2008. 86:64-72. doi:10.2527/jas.2007-0399
© 2008 American Society of Animal Science
ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Ectopic expression of porcine peroxisome proliferator-activated receptor 
regulates adipogenesis in mouse myoblasts1
Y. H. Yu,
S. C. Wu,
W. T. K. Cheng,
H. J. Mersmann2 and
S. T. Ding3
Department of Animal Science and Technology/Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan
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Abstract
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Peroxisome proliferator-activated receptor 
(PPAR) plays a critical role in regulating adipogenesis. The expression of peroxisome proliferator-activated receptor 
(PPAR) precedes that of PPAR during adipocyte differentiation in rodents. The current experiment was designed to study the function of porcine PPAR and the interaction of PPAR and PPAR in adipocyte differentiation. Inhibition of myogenesis was observed in mouse myoblasts expressing porcine PPAR, similar to myoblasts expressing PPAR. Treatment of myoblasts expressing PPAR with ligands for both PPAR and PPAR enhanced lipogenesis and adipogenesis to a greater extent than treatment with a PPAR ligand alone, suggesting that both genes were involved in regulating lipogenesis and adipogenesis. The ability to transdifferentiate myoblasts into adipocytes was decreased in myoblasts coexpressing PPAR with either wild type or mutated PPAR (Ser 112 was mutated to Ala; the mutated PPAR is more active than the wild type) compared with myoblasts expressing PPAR alone. Adipocyte differentiation in myoblasts coexpressing PPAR and mutated PPAR was greater than in myoblasts coexpressing PPAR and wild type PPAR, confirming that Ser 112 is important for the function of PPAR. Taken together, our results demonstrate that overexpression of PPAR inhibits myotube formation and also enhances adipocyte differentiation. However, the complexity and interaction of PPAR and PPAR in adipogenesis are not clearly understood.
Key Words: adipocyte differentiation • myogenin • peroxisome proliferator-activated receptor • peroxisome proliferator-activated receptor • pig
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INTRODUCTION
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In rodents, peroxisome proliferator-activated receptor (PPAR) is widely expressed in several tissues, including adipose tissue, intestine, skeletal muscle, lung, and heart (Amri et al., 1995
). The expression of PPAR in proliferating preadipocytes is undetectable but increases gradually during rodent adipocyte differentiation; the expression of PPAR precedes that of peroxisome proliferator-activated receptor (PPAR) in differentiating rodent adipocytes (Amri et al., 1995). Addition of a long-chain fatty acid, palmitic acid, to preadipocytes overexpressing PPAR increases adipogenesis (Bastie et al., 2000). However, ectopic expression of PPAR in fibroblasts does not induce adipogenesis, even after addition of a long-chain fatty acid, whereas adipogenesis is stimulated after addition of a PPAR ligand (Bastie et al., 1999). A contrasting observation is that lipid accumulation is not observed in fibroblasts expressing PPAR even in the presence of PPAR and PPAR ligands (Brun et al., 1996). These results suggest that PPAR may have a role during adipocyte differentiation, but the precise function of PPAR in rodent adipocyte differentiation is not well defined. Moreover, there is no evidence to show the function of porcine PPAR in adipocyte differentiation. Thus, identification of the role of porcine PPAR in regulating adipocyte differentiation is needed.
In previous studies, we demonstrated that ectopic expression of porcine PPAR induces adipogenesis in myoblasts after addition of a PPAR ligand (Yu et al., 2006). Whether PPAR can enhance the effect of PPAR on porcine adipocyte differentiation is not known. Therefore, we created C2C12 myoblasts expressing porcine PPAR, or myoblasts coexpressing PPAR with either wild type or mutated (Ser 112 mutated to Ala) porcine PPAR to test the hypothesis that PPAR and PPAR interact to regulate adipocyte differentiation.
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MATERIALS AND METHODS
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The animal protocol was approved by the Animal Care and Use Committee of the National Taiwan University.
Gene Construction
Two 2-mo-old crossbred pigs were killed by electrical stunning combined with exsanguination for gene cloning (Liu et al., 2005). Pig adipose tissue RNA was extracted with guanidinium-phenol-chloroform following the procedure described by Chomczynski and Sacchi (1987), with modifications by Hsu and Ding (2003). Reverse transcribed cDNA was used for cloning porcine full-length PPAR (GenBank accession no. DQ437886). The cDNA was inserted into a mammalian expression vector, according to the directions from the supplier (pIRESpuro3, Clontech, Mountain View, CA), to drive porcine PPAR expression. All the cloned plasmids in the current study were sequenced, and the sequences were confirmed.
Stable Cell Lines
To establish cells stably expressing porcine PPAR, C2C12 myoblasts (ATCC CRL-1772, ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, as previously described (Yu et al., 2006). At 80% confluence, the cells were transfected with empty vector or vector containing PPAR by lipofection according to directions from the supplier (Lipofectamine 2000, Invitrogen, Carlsbad, CA). In addition, stable cell lines expressing either wild type PPAR or mutated PPAR (Yu et al., 2006) were also stably transfected with porcine PPAR. Transfected cells were selected with puromycin for at least 1 mo. After antibiotic selection, single colonies were isolated with a cloning cylinder (Sigma, St. Louis, MO) for further propagation.
Induction of Adipogenesis in Myoblasts
All reagents were purchased from Sigma-Aldrich (St. Louis, MO). For adipogenesis, confluent genetically modified cells were cultured in adipogenic differentiation medium (Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 1 µM dexamethasone, and 5 µg/mL of insulin) and with or without 1 µM rosiglitazone, an antidiabetic drug with high ligand-binding activity for PPAR, or 1 µM L165041, a leukotriene antagonist with high ligand-binding activity for PPAR. All PPAR ligands were dissolved in dimethyl sulfoxide for the experiment. The medium was changed every 2 d. Our previous data showed that after 10 d of treatment, the PPAR-transfected C2C12 cells began to differentiate into adipocytes (Yu et al., 2006). Therefore, cells were stained with oil-red O to measure triacylglycerol deposition after 10 d of culture, and intracellular lipids were quantified by extraction of oil-red O dye. Details for the procedure were described by Ramirez-Zacarias et al. (1992). The number of myotubes per square millimeter in the culture dishes was determined.
Northern Blot
The RNA was extracted and separated by electrophoresis and blotted to nylon membranes. The membrane was prehybridized at 42°C and then hybridized with isotope-labeled complementary DNA probes. The probes for PPAR and glyceraldehyde-3-phosphate de-hydrogenase were generated by PCR using primer sequences described previously (Yu et al., 2006). The primer sequences for the PPAR probe were sense 5'-GTACGAGAAGTGTGAGCGGA-3' and antisense 5'-GAGGAAGAAGTGGTCGAAGC-3' from the sequence in GenBank accession no. DQ437886. Hybridization results were quantified by phosphor image analysis (ImageQuant TL v2005 software, GE Health). The densitometric value for an individual transcript in a sample lane was normalized to the densitometric value for the glyceraldehyde-3-phosphate dehydrogenase mRNA in the same lane. Details for all of these procedures were described previously (Chen et al., 2006; Wang et al., 2006).
Statistical Analysis
The treatment effects were analyzed by using AN-OVA to determine the main effects of the PPAR and PPAR in the presence or absence of their ligands. Duncan’s new multiple-range test was used to evaluate differences among means (SAS Inst. Inc., Cary, NC). A significant difference indicates that the P-value was 
0.05.
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RESULTS
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Ectopic Expression of Porcine PPAR in Myoblasts
Endogenous PPAR mRNA expression in C2C12 myoblasts carrying empty vector was weak, whereas a high level of PPAR mRNA expression was detected in myoblasts transfected with either PPAR alone or PPAR and PPAR (Figure 1).
PPAR Facilitates Adipogenesis
To determine the degree of triacylglycerol accumulation, cells were stained with oil-red O and photographed after 10 d of treatment. In myoblasts transfected with empty vector and exposed to adipogenic medium, there were considerable myocyte differentiation and some myotube formation but no evidence of adipogenesis (Figure 2A). Myogenesis, but not adipogenesis, was evident in these cells even when a PPAR ligand, a PPAR ligand, or both the PPAR and PPAR ligands were present (Figure 2B to 2D). Myotube formation was inhibited in myoblasts expressing porcine PPAR (Figure 2E to 2H). Addition of the PPAR ligand, rosiglitazone, to the adipogenic medium brought about lipid-droplet formation (positive oil-red O staining) in cells expressing PPAR (Figure 2F). However, in the presence of the PPAR ligand, L165041, no adipogenesis was observed (Figure 2G). Although treatment with the PPAR ligand alone was not sufficient to trigger transdifferentiation of myoblasts into adipocytes, exposure to both the PPAR and PPAR ligands enhanced lipid deposition in myoblasts expressing PPAR (Figure 2H). The number of myotubes in myoblasts transfected with empty vector was greater than in cells with PPAR (Figure 3A). The addition of the PPAR ligand or both the PPAR and PPAR ligands increased the intracellular triacylglycerol accumulation in cells expressing PPAR (Figure 3B).
The mRNA level of the adipogenic marker gene, adipocyte fatty acid-binding protein (aP2), was low in myoblasts containing empty vector even in the presence of PPAR ligand, PPAR ligand, or both the PPAR and PPAR ligands (Figure 4A). Myoblasts expressing PPAR also had a low level of aP2 mRNA without or with addition of the PPAR ligand (Figure 4A). However, the level of aP2 mRNA was greatly increased in myoblasts expressing PPAR when the PPAR ligand was added to the adipogenic medium; the addition of both the PPAR and PPAR ligands increased the aP2 mRNA even further (Figure 4A). The mRNA for another adipogenic marker gene, lipoprotein lipase (LPL), was not detected in myoblasts carrying empty vector, with or without the PPAR ligand, PPAR ligand, or PPAR and PPAR ligands (Figure 4B). In myoblasts transfected with PPAR, addition of the PPAR ligand or both the PPAR and PPAR ligands caused a large increase in the LPL mRNA (Figure 4B). The results for aP2 and LPL mRNA were similar. Our results demonstrated that in myoblasts expressing porcine PPAR with or without its ligand, adipogenesis (as assessed by morphology, triacylglycerol accumulation, or mRNA levels for aP2 and LPL) was not stimulated. In myoblasts expressing PPAR, lipid deposition and adipogenic gene expression were increased when the adipogenic medium contained the PPAR ligand; adipogensis was further enhanced in the presence of both the PPAR and PPAR ligands.
The mRNA levels for the myogenic marker genes, myogenin and myogenic regulatory factor-4 (MRF4), were the same in myoblasts containing empty vector when grown in adipogenic medium with or without the various PPAR ligands (Figure 5A and 5B). In contrast, myoblasts expressing PPAR and grown in adipogenic medium had significantly decreased myogenin and MRF4 mRNA levels (Figure 5A and 5B). These results demonstrated that porcine PPAR had the ability to block the myogenic program even when no exogenous PPAR ligand was added.
The observation that adipogenesis in myoblasts expressing PPAR was stimulated by a PPAR ligand, but not by a PPAR ligand, suggests that the adipogenic effect was mediated by endogenous PPAR. Consequently, we measured endogenous PPAR mRNA levels in myoblasts expressing PPAR during exposure to adipogenic medium. The PPAR mRNA levels were low and did not change with the time of exposure to adipogenic medium in myoblasts expressing PPAR (Figure 6). After addition of the PPAR ligand (L165041) to the adipogenic medium, the PPAR mRNA level increased and continued to increase to d 9 (Figure 6). These results demonstrated that porcine PPAR with its ligand induced expression of endogenous PPAR. Adipogenesis was not observed unless a PPAR ligand was present (Figures 2
, 3, and 4).
Coexpression of PPAR and PPAR in Myoblasts Decreases Adipogenic Gene and Myogenic Gene Expression
In a previous study (Yu et al., 2006), we demonstrated that porcine PPAR directly regulates adipogenesis in myoblasts. In the current study, we demonstrated that porcine PPAR had a role in adipogenesis. Therefore, we tested whether expression of both porcine PPAR and PPAR in myoblasts could enhance transdifferentiation of myoblasts into adipocytes compared with expression of PPAR or PPAR alone. In myoblasts transfected with either wild type porcine PPAR or mutated PPAR (phosphorylation of PPAR diminishes its activity so that the mutation of Ser 112 to Ala decreases phosphorylation to produce a more active PPAR), the mRNA for the adipogenic marker genes, aP2 and LPL, were increased when a PPAR ligand (rosiglitazone) was added (Figure 7A and 7B). Addition of both the PPAR and PPAR ligands further enhanced the levels of aP2 and LPL mRNA (Figure 7A and 7B). Although the myoblasts transfected with both PPAR and PPAR had increased mRNA for aP2 and LPL when a PPAR ligand was added and even greater mRNA levels when both a PPAR and a PPAR ligand were added, the aP2 and LPL mRNA levels were considerably reduced compared with cells transfected only with wild type or mutated PPAR. The myogenic differentiation genes, myogenin and especially MRF4, were expressed at lower levels in myoblasts containing both PPAR and PPAR compared with myoblasts containing only PPAR (Figure 8A and 8B).
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DISCUSSION
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The PPAR is a member of the 48 nuclear receptors with 4 common functional domains including transcription activation, DNA binding, hinge, and ligand binding, identified in the human and mouse genome that regulate gene expression related to lipid metabolism (reviewed by Khan and Vanden Heuvel, 2003). Three PPAR isoforms (
, , and ) have been described (Kota et al., 2005). Each isoform has a different role in lipid metabolism. In rodents, PPAR is highly expressed in liver, kidney, and heart (Braissant et al., 1996). In pigs, PPAR is highly expressed in adipose tissue, in addition to liver and skeletal muscle (Ding et al., 2000). Several key enzymes involved in peroxisomal β-oxidation are regulated by PPAR, for example, acyl-CoA oxidase (Dreyer et al., 1992). Fatty acid oxidation disorders are found in the PPAR-null mouse during fasting (Leone et al., 1999), indicating that PPAR can modulate fatty acid catabolism. Adipose tissue is the major site for expression of PPAR, especially PPAR2 (Tontonoz et al., 1994). Expression of PPAR, especially PPAR1, is also observed in several tissues (Zhu et al., 1995). The PPAR2 is an essential regulator to initiate adipocyte differentiation in rodents (Rosen et al., 1999) and pigs (Yu et al., 2006). The PPAR, also called PPARβ or NUC-1, is ubiquitously expressed and is abundant in adipose tissue, muscle, intestine, brain, and heart (Amri et al., 1995). Overexpression of PPAR in Ob1771 preadipocytes increases adipocyte differentiation when a PPAR ligand is present (Bastie et al., 2000). Fat mass is reduced in PPAR-null mice (Barak et al., 2002). In addition, PPAR also regulates lipid catabolism (Holst et al., 2003). It has been demonstrated that several fatty acids (i.e., palmitic acid and eicosapentaenoic acid) could activate PPAR (Amri et al., 1995; Forman et al., 1997). The leukotriene antagonist L165041 is a synthetic ligand that has high-affinity ligand binding and specificity for PPAR compared with PPAR or PPAR (Willson et al., 2000). Similar to rodents, porcine PPAR is expressed in several tissues (Lord et al., 2006). The AA sequence of porcine PPAR is 89 and 90% homologous to that of the rat and mouse, respectively (Lord et al., 2006). The role of porcine PPAR in lipid metabolism has not been studied.
Lipid accumulation and PPAR-mediated adipogenic genes are decreased in PPAR-null adipocytes (Matsusue et al., 2004). In addition, ectopic expression of PPAR in mouse fibroblasts promotes adipogenic gene expression when a long-chain fatty acid is added, but these cells do not undergo adipogenesis unless a PPAR ligand is added (Bastie et al., 1999). In our studies, no lipid accumulation was observed in myoblasts expressing porcine PPAR with or without the PPAR ligand. Similar to the results of Bastie et al. (1999), addition of a PPAR ligand to the adipogenic medium caused these myoblasts to transdifferentiate into adipocytes. These results indicate that PPAR alone is not able to trigger lipid deposition and that the presence of a PPAR ligand is essential to trigger the adipogenic program in either fibroblasts or myoblasts. In the current study, we demonstrated that myoblasts carrying PPAR in the presence of adipogenic medium plus a PPAR ligand had a continuously increased content of endogenous PPAR mRNA. Adipogenesis was not increased under these circumstances. Speculatively, the PPAR ligand, L165041, did not activate the induced PPAR and there was a less than optimal concentration of an endogenous PPAR ligand present to stimulate adipogenesis. Myoblasts expressing PPAR, but with no exogenous PPAR ligand added to the adipogenic medium, had no observable adipogenesis and did not have an increased expression of endogenous PPAR suggesting there was insufficient endogenous PPAR ligand present. Addition of an exogenous PPAR ligand to the myoblasts expressing PPAR caused a marked increase in adipogenesis, suggesting that the PPAR ligand cross-activated the PPAR (to induce endogenous PPAR) or that these circumstances promoted the production of an endogenous PPAR ligand. Numerous PPAR ligands can activate more than one receptor isoform (Berger et al., 1999). When both a PPAR and PPAR ligand were added to the adipogenic medium, the adipogenic effect was enhanced, compared with addition of the PPAR ligand alone. These findings demonstrated that the high level of lipid deposition in myoblasts expressing PPAR and grown in adipogenic medium plus a PPAR ligand results from the induction of endogenous PPAR by PPAR with adipogenesis directly stimulated by the endogenous PPAR activated by the exogenous PPAR ligand. Therefore, PPAR seems to play a facilitating role during adipogenesis.
In myoblasts transfected with the wild type or mutated PPAR, addition of a PPAR ligand to the adipogenic medium enhanced adipogenesis, as observed previously (Yu et al., 2006). The mutated PPAR with Ser 112 changed to Ala has a reduced capacity to be phosphorylated, with concomitant inactivation. Addition of both a PPAR and PPAR ligand further enhanced adipogenesis. Perhaps the exogenous PPAR ligand activated endogenous PPAR (to promote adipogenesis by increasing the pool of PPAR through induction of endogenous PPAR). Because the cells expressing PPAR in the presence of the exogenous PPAR ligand do not have increased adipogenesis, we concluded that the PPAR ligand does not activate PPAR.
It has been demonstrated that PPAR represses PPAR and PPAR target gene expression in fibroblasts carrying both PPAR and PPAR or PPAR (Shi et al., 2002). We observed a similar phenomenon in myoblasts coexpressing PPAR and either wild type PPAR or mutated PPAR; the PPAR target gene mRNA (i.e., aP2 and LPL mRNA) were reduced. This result implies that PPAR restricts expression of PPAR during adipogenesis. In support of this concept, disruption of the PPAR DNA-binding domain significantly overcomes the PPAR inhibition in fibroblasts expressing both PPAR and PPAR or PPAR (Shi et al., 2002); the results indicate that the decreased PPAR target gene expression is due to PPAR binding to the PPAR response element.
Nuclear receptor corepressor, the silencing mediator of retinoid and thyroid hormone and Cyclin D1, has an inhibitory effect on PPAR function (Fu et al., 2005; Yu et al., 2005). There is no direct evidence to demonstrate that PPAR regulates PPAR repressors during adipogenesis. However, it is possible that PPAR may recruit repressors to modulate PPAR function. Retinoid X receptor (RXR) expression does not appear to be involved in PPAR-mediated repression of PPAR or PPAR target gene expression (Shi et al., 2002). Regardless, PPAR may compete with PPAR for heterodimerization with RXR to partially reduce the amount of functional PPAR-RXR required for adipogenesis.
Porcine PPAR had an inhibitory role on myogenesis whether PPAR ligands were present or not. We previously observed that myogenesis was inhibited in myoblasts expressing porcine PPAR (Yu et al., 2006). Furthermore, myogenesis was decreased in myoblasts ectopically expressing both PPAR and PPAR. These results indicate that PPAR and PPAR have a similar ability to block the myogenic program of myoblasts. Although we observed a significant decrease in myogenesis in myoblasts expressing either PPAR or PPAR, the inhibition of PPAR on myogenesis did not seem to be necessary for promoting adipogenesis of myoblasts.
In conclusion, we demonstrated that in myoblasts transfected with porcine PPAR, adipogenesis was promoted in the presence of a PPAR ligand. Furthermore, myogenesis was blocked. In addition, we showed that PPAR alone with its ligand modulates adipogenic genes via expression of endogenous PPAR. The observation that stable transformation of myoblasts with both PPAR and PPAR suppressed adipogenesis compared with cells expressing only PPAR (with increased expression of endogenous PPAR) indicates that the complexities and interactions of PPAR and PPAR in adipogenesis are not completely understood.
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Footnotes
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1 The project was funded in part by the National Science Council of Taiwan. 
2 Visiting professor at National Taiwan University.
3 Corresponding author: sding{at}ntu.edu.tw
Received for publication July 4, 2007.
Accepted for publication September 3, 2007.
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Abstract
INTRODUCTION
