J. Anim Sci. 2006. 84:2655-2665. doi:10.2527/jas.2005-645
© 2006 American Society of Animal Science
ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Porcine peroxisome proliferator-activated receptor 
induces transdifferentiation of myocytes into adipocytes1
Y. H. Yu,
B. H. Liu,
H. J. Mersmann2 and
S. T. Ding3
Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan
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Abstract
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Peroxisome proliferator-activated receptor 
2 (PPAR) is a nuclear transcription factor that regulates adipocyte differentiation and lipogenic genes during adipogenesis. The activity of rodent PPAR is regulated by phosphorylation of serine 112. The current experiment was designed to study the ability of porcine PPAR to stimulate transdifferentiation of myoblasts to adipocytes by overexpressing wild-type PPAR or mutated PPAR (serine 112 was mutated to alanine) in mouse myoblast cells. The expression of adipogenic marker genes (adipocyte fatty acid binding protein, lipoprotein lipase, and glycerol-3 phosphate dehydrogenase) in cells stably expressing mutated porcine PPAR was greater than in cells with wild-type PPAR, indicating that the mutated PPAR has greater adipogenic capability than the wild-type PPAR. Under treatment with a ligand, both wild-type and mutant porcine PPAR-expressing C2C12 myoblasts differentiated into adipocytes in 10 d. The expression of myogenic marker genes (myogenin, myogenic regulatory factor-4) was suppressed in cells transfected with the mutated PPAR or wild-type PPAR. Moreover, wild-type and mutant PPAR were able to inhibit myogenesis without addition of a ligand. Our results suggest that porcine wild-type PPAR and mutated PPAR can both convert myoblast cells into adipocytes, and also that the ability to transdifferentiate was greater in cells containing the mutated PPAR than in cells containing the wild-type PPAR. Therefore, the existence of serine 112 in PPAR may have a role in regulating adipocyte differentiation.
Key Words: adipocyte differentiation • adipocyte gene • myoblast transdifferentiation • myocyte gene • peroxisome proliferator-activated receptor
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INTRODUCTION
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The peroxisome proliferator-activated receptor 2 (PPAR) is a critical transcription factor in the regulation of adipogenesis. Ectopic expression of rodent PPAR in fibroblasts stimulates adipocyte development (Tontonoz et al., 1994
). In addition, expression of rodent PPAR and the CCAAT/enhancer-binding protein 
(C/EBP) in myoblasts activates an adipogenic program in this cell line (Hu et al., 1995). The regulation of PPAR activity is posttranslationally modified. Phosphorylation of rodent PPAR serine 112 by mitogen-activated protein (MAP) kinase decreases the ability of PPAR to regulate adipogenic genes and decreases differentiation (Hu et al., 1996). In NIH 3T3 cells, replacement of rodent PPAR serine 112 with alanine promotes the function of PPAR during adipogenesis and enhances the ligand sensitivity through prevention of MAP kinase phosphorylation (Hu et al., 1996).
Thiazolidinediones (TZD), compounds used to treat type 2 diabetes, are ligands for PPAR (Kletzein et al., 1992). The TZD act as adipogenic agents in porcine preadipocytes, suggesting they are ligands for porcine PPAR (Tchoukalova et al., 2000). Although the expression of PPAR is positively associated with porcine adipocyte differentiation (Ding et al., 1999), there is no direct evidence to demonstrate the function of porcine PPAR on adipocyte differentiation. Intramuscular fat is not abundant in porcine muscle. Thus, it is of interest to increase the number of intramuscular adipocytes. In this study, we transfected the myogenic C2C12 myoblast cell line with wild-type or mutated porcine PPAR (ser 112
ala). Upon addition of a PPAR ligand, transfected cells with wild-type PPAR or mutated PPAR were transdifferentiated from a myogenic to an adipogenic type.
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MATERIALS AND METHODS
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Full Length cDNA Cloning and Gene Construction
The animal protocol was approved by the Animal Care and Use Committee of the National Taiwan University. Two 2-mo-old crossbred pigs were killed by electrocution combined with exsanguination for gene cloning and gene expression studies (Liu et al., 2005). Longissimus muscle and s.c. adipose tissue were obtained, and RNA was extracted with guanidinium-phenol-chloroform (Chomczynski and Sacchi, 1987) using modifications by Hsu and Ding (2003). Total RNA was reverse transcribed using a kit, SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA). First strand cDNA from pig adipose tissue was used for cloning PPAR2 (Accession No. AF103946). The point-mutated PPAR (ser 112ala), which resulted in a nonfunctional phosphorylation site, was created by primer design. The PCR products were cloned into a mammalian expression vector with a CMV promoter (pIRES-EGFP, Clontech, Mountain View, CA) to drive the expression of wild-type PPAR or mutated PPAR. Sequences of these recombinant molecules were determined and confirmed.
Establishment of Cell Lines Stably Expressing Pig PPAR
The C2C12 myoblasts (CRL-1772, ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C in an atmosphere of 5% CO2 in air. After confluence, C2C12 myoblast cells differentiated into mature myotubes when the serum concentration was lowered by replacing the 10% FBS with 2% horse serum.
The empty mammalian expression vector (CV) or vector containing wild-type PPAR or mutated PPAR was transfected into C2C12 myoblasts by lipofection (Fugene 6, Roche Applied Science, Indianapolis, IN). Cell lines stably expressing CV or PPAR were established by antibiotic selection using G418. In this system, antibiotic resistance is an indication of successful integration of foreign genes into the genome of the cells. After transfection with individual plasmids, cells were maintained in nonselective medium for 2 d, after which the nonselective medium was replaced with selective medium containing the antibiotic. The selection was for at least 1 mo. Dead cells were eliminated through frequent replacement of the selective medium, until distinct colonies could be visualized under the selective-medium environment. Individual colonies were isolated to culture cells for further propagation.
Induction of Myocyte Transdifferentiation
After the cell lines were established, the cells were cultured without selection medium and allowed to propagate to 80% confluence in DMEM with 10% FBS. Confluent cells were then cultured in adipogenic differentiation medium [DMEM containing 10% FBS, 1 µM dexamethasone, and 5 µg/mL insulin with or without 1 µM rosiglitazone, a PPAR ligand]. For myogenic differentiation, cells were cultured in DMEM plus 2% horse serum, with or without 1 µM PPAR ligand. Cells were cultured in myogenic or adipogenic medium for 10 d, with a medium change every 2 d. The adipogenic and myogenic media ± rosiglitazone, were used to assess the capacity of C2C12 myoblasts containing wild-type or mutated PPAR to modify adipogenesis and myogenesis. Each experiment was repeated 3 times.
After 10 d of culture, cells on the plates were stained with Oil Red O to measure the degree of adipocyte differentiation (Ramirez-Zacarias et al., 1992). Cellular RNA was extracted to determine the mRNA concentrations for several genes whose expression increases during adipocyte differentiation: PPAR, lipoprotein lipase (LPL; an early marker), adipocyte fatty acid-binding protein (aP2; a late marker), and glycerol-3 phosphate dehydrogenase (GPDH; a marker for triacylglycerol synthesis activity). The mRNA for genes representating myoblast differentiation were also measured: myoblast determination protein-1 (MyoD; an early marker), myogenic factor-5 (Myf5; an early marker), myogenin (a late marker), and myogenic regulatory factor-4 (MRF4; a late marker).
Extraction of RNA
Total RNA was extracted for northern analysis. The quality of the RNA was monitored by examination of the 18S and 28S ribosomal RNA bands after electrophoresis. The RNA was quantified by spectrophotometry at 260 nm and stored at –70°C.
Northern Analysis
Total RNA (20 µg of each sample) was electrophoresed and transferred to nylon membranes. The probes for the genes measured were generated by PCR using the primer pairs listed in Table 1. The membrane was prehybridized at 42°C in UltraHyb (Ambion, Austin, TX) for 1 h, and then the denatured cDNA probe (95°C for 5 min) was added at a concentration of 1 pM and allowed to hybridize with the targeted gene transcripts overnight at 42°C. Hybridization was quantified by phosphor-image analysis as previously described (Hsu et al., 2004; Wang et al., 2006). The densitometric value for an individual transcript in a sample lane was normalized to the densitometric value for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the same lane.
Statistical Analysis
The treatment effects were analyzed using an AN-OVA procedure to determine the main effects of the form of PPAR and presence or absence of a ligand. Duncan’s new multiple range test was used to evaluate differences among means (SAS Inst. Inc., Cary, NC). A significant difference indicates that P value is not greater than 0.05.
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RESULTS
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Ectopic Expression of Wild-Type PPAR and Mutated PPAR in C2C12 Myoblasts
Expression of pig PPAR in adipose tissue was robust, whereas expression in muscle was weak (Figure 1). No PPAR was detected in the RNA from un-transfected C2C12 myoblasts (Figure 1). However, C2C12 myoblasts transfected with wild-type porcine PPAR or mutated porcine PPAR expressed a high level of PPAR mRNA (Figure 1).
Wild-Type PPAR and Mutated PPAR Trigger Transdifferentiation in Adipogenic Medium
The stably transformed porcine PPAR or mutated PPAR increased the expression of aP2 and LPL, 2 PPAR targeting genes (Figure 2). Two empty vector containing C2C12 clones (CV1 and CV2) expressed aP2, LPL, MyoD, and myogenin (myogenic markers) in the same manner as in the normal C2C12 cells, indicating that the insertion of the vector does not have an effect on the expression of adipogenic and myogenic genes in these cells. The addition of TZD did not affect the expression of adipogenic or myogenic genes in the CV1 and CV2 (Figure 2). Normal C2C12 cells differentiate to multinucleated myotubes in adipogenic medium (Figure 3, panel A). The presence or absence of rosiglitazone in adipogenic medium had no effects on myogenesis (Figure 3, panel A vs. D). Rosiglitazone at 1 µM did not trigger myoblast transdifferentiation into adipocytes. In contrast, myoblasts expressing native PPAR or mutated PPAR were maintained but did not progress to myotubes (Figure 3, panels B and C). This observation demonstrated that porcine PPAR was able to block the myogenic program without exogenous ligand activation. However, after addition of rosiglitazone to the adipogenic differentiation medium for 10 d, lipid droplets were visualized in myoblasts expressing wild-type PPAR and mutated PPAR (Figure 3, panels E and F). To determine the degree of accumulation of intracellular triacylglycerol, cells were stained with Oil-Red-O and photographed on d 10 (Figure 3, panels G to L). After Oil-Red-O staining, myoblasts expressing wild-type PPAR or mutated PPAR displayed a low degree of triacylglycerol accumulation when no ligand was added to the adipogenic medium (Figure 3, panels H and I). However, after addition of the ligand to adipogenic medium, high levels of triacylglycerol were observed in wild-type PPAR and mutated PPAR C2C12 myoblasts (Figure 3, panels K and L).
Normal C2C12 myoblasts in adipogenic medium had a low level of PPAR mRNA expression (Figure 4, panel A). The PPAR mRNA expression may result from the medium insulin and dexamethasone, but the expression was not great enough to trigger the myoblast transdifferentiation even in the presence of rosiglitazone. The PPAR mRNA expression in myoblasts containing wild-type PPAR or mutated PPAR was the same and at a high level (Figure 4, panel A). These levels were enough to cause the expression of downstream genes for adipogenesis, especially in the presence of the PPAR ligand, rosiglitazone. Lipoprotein lipase could not be detected in normal C2C12 (Figure 4, panel B). However, high levels of LPL expression were obtained in cells ectopically expressing PPAR under rosiglitazone stimulation (Figure 4, panel B). Similar results (Figure 4, panels C and D) were obtained for other PPAR activated genes, aP2 and GPDH. Further experiments demonstrated that the effectiveness of increasing the expression of PPAR targeting genes (aP2 and LPL) was greater by mutated PPAR compared with the wild-type PPAR under several concentrations of its ligand (Figure 5). These results demonstrated that exogenous porcine PPAR could cause expression of adipogensis-related genes to trigger the adipogenic program in C2C12 myoblasts.
Myogenic gene, MyoD, was expressed in all cell types at an equivalent level in the presence or absence of ligand (Figure 6, panel A). However, another early expressed myogenic marker, Myf5, was expressed at a greater level in cells transfected with wild-type PPAR or mutated PPAR compared with control cells; addition of rosiglitazone had no effect (Figure 6, panel B). The mRNA for myogenin and MRF4, genes representing later stages of myoblast differentiation, were expressed at a lower level in C2C12 cells expressing the wild-type PPAR or the mutated PPAR compared with normal C2C12 (Figure 6, panels C and D). Our experiments demonstrated that PPAR could down-regulate myogenic differentiation genes and inhibit myogenesis in adipogenic medium whether rosiglitazone was present or not.
Wild-Type PPAR and Mutated PPAR Suppressed Myogenesis
Normal C2C12 myoblasts differentiate very well to form myotubes under a 2% horse serum treatment (Figure 7, panel A). Addition of the PPAR-ligand to the myogenic medium had no effect on myogenic differention (Figure 7, panel D). In contrast, cells expressing wild-type PPAR or mutated PPAR maintained an undifferentiated state, and myotubes were rarely visualized in myogenic medium (Figure 7, panels B and C). Addition of rosiglitazone to myogenic medium triggered a small degree of readily visualized lipid-droplet formation in both kinds of genetically modified cells (Figure 7, panels E and F).
The expression of PPAR was greater in C2C12 cells expressing wild-type or mutated PPAR compared with normal C2C12 (Figure 8, panel A). The adipogenic gene LPL was not expressed in normal myoblasts but was observed in myoblasts transfected with PPAR (Figure 8, panel B). Addition of a PPAR-ligand, rosiglitazone further enhanced adipogenesis, as indicated by an increase number of lipid-containing cells (Figure 8, panels E and F) and increased LPL mRNA (Figure 8, panel B). The C2C12 cells expressing wild-type or mutated PPAR both expressed aP2 and GPDH when the PPAR-ligand was added (Figure 8, panels C and D). Thus, increased adipogenic gene expression was detected before extensive lipid deposition. The cells expressing the mutated PPAR had increased levels of mRNA for adipogenic genes compared with cells expressing the wild-type PPAR (Figure 8, panels B, C, and D).
The expression of MyoD was the same in normal and genetically modified C2C12 cells, maintained in myogenic medium (Figure 9, panel A). Another myogenesis early expressed gene, Myf5, was more highly expressed in both types of genetically modified C2C12 myoblasts than in normal C2C12 (Figure 9, panel B). Expression of the myogenic terminal differentiation genes, myogenin and MRF4, was decreased by the presence of PPAR (Figure 9, panels C and D). Addition of the PPAR-ligand did not further decrease expression of these genes.
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DISCUSSION
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Preadipocyte and myoblast lineages are derived from the same multipotent mesodermal progenitor (Grigoriadis et al., 1988). After determination, preadipocytes and myoblasts undergo terminal differentiation and develop into functional cells. Differentiation of preadipocytes into adipocytes is regulated by several transcription factors, including PPAR, C/EBP, and sterol regulatory element binding protein-1c (SREBP-1c or ADD1). During rodent adipogenesis, PPAR is an early expressed transcription factor that stimulates the process of adipocyte differentiation in vitro and in vivo (Rosen et al., 1999). Several adipogenesis-related genes are modulated by binding the ligand activated PPAR to their PPAR-response elements. For example, aP2 (Tontonoz et al., 1994) and phosphoenolpyruvate carboxykinase (Tontonoz et al., 1995) are regulated by PPAR. Therefore, the primary transcription factor to stimulate rodent adipogenesis is PPAR (Rosen et al., 1999).
The differentiation of myoblasts and preadipocytes involves different transcription factor programs. In myogenesis, this process is regulated by a family of basic helix-loop-helix transcription factors, including MyoD, Myf5, myogenin, and MRF4. The MyoD and Myf5 are expressed at early stages and participate in myoblast determination, whereas myogenin and MRF4 are expressed at later stages and promote myotube formation in terminal differentiation (Weintraub et al., 1989; Emerson, 1993). In recent years, mouse C2C12 and G8 myoblast cell lines have been used to study the myogenic program. In addition to myogenesis, C2C12 myoblasts can differentiate into osteoblasts and adipocytes under appropriate culture conditions (Katagiri et al., 1994; Teboul et al., 1995; Nishimura et al., 1998). Furthermore, ectopic expression of rodent PPAR and C/EBP in G8 myoblasts induces myoblast conversion to adipocytes (Hu et al., 1995).
In rodents, the PPAR serine 112 can be phosphorylated by MAP kinase during preadipocyte proliferation, and the phosphorylated PPAR has reduced transcriptional regulatory activity (Hu et al., 1996; Shao et al., 1998). In the current study, the mutation of serine 112 in the porcine PPAR seems to enhance the ability of PPAR to regulate transcription of its targeting genes. However, a lack of phosphorylation information for the porcine PPAR limits the ability to speculate the precise mechanism. Regardless of the precise mechanism, the mutated PPAR has greater ability to promote adipogenesis in our C2C12 myoblasts than does the wild-type PPAR. The results also support a model in which decreased phosphorylation of mutated PPAR enhances transcriptional activity to drive adipogenesis.
The TZD enhance the sensitivity of tissues to insulin and are widely used to treat type-2 diabetes. Preadipocytes treated with TZD have increased expression of adipogenesis-related genes and differentiation (Kletzien et al., 1992). It has been suggested that 5 µM rosiglitazone activates adipogenic genes in mouse C2C12 myoblasts to trigger the conversion to adipocytes (Teboul et al., 1995), although the rat L6 myoblast cell line did not trandifferentiate into adipocytes in the presence of TZD (Hammarstedt and Smith, 2003). In our studies, addition of 1 µM rosiglitazone to C2C12 myoblasts without the transfected PPAR had no effect on transdifferentiation in adipogenic or myogenic medium, suggesting that 1 µM rosiglitazone was not enough to trigger the adipogenic program of myoblasts without the expression of exogenous PPAR.
Expression of PPAR and C/EBP in G8 myoblasts causes transdifferentiation into adipocytes (Hu et al., 1995). In addition, C2C12 myoblasts transfected with PPAR
had increased endogenous PPAR and conversion to adipocytes under TZD stimulation (Holst et al., 2003). These results imply that ectopic expression of PPAR with its ligand is able to enhance myocyte transdifferentiation to adipocytes. In the current studies, the addition of rosiglitazone to cells expressing porcine PPAR increased the expression of adipogenic genes, i.e., LPL, aP2, and GPDH. In adipogenic medium, the mutated PPAR had an increased capacity to enhance adipogenesis compared with the wild-type PPAR only in the presence of the ligand. The strength of the adipogenic outcome in cells transfected with either PPAR is indicated by the presence of a few lipid containing cells and increased LPL mRNA even when the myoblasts were cultured in myogenic medium.
The terminal myogenic differentiation genes, myogenin and MRF4, were reduced in cells containing either PPAR type. During myogenesis, the MyoD expression is present at a steady state level, whereas the expression of Myf5 increased and peaked at d 4 then reduced until d 10 (Dedieu et al., 2002). We observed the same expression of MyoD in all cells. However, the reason for the increased Myf5 expression in genetically modified cells compared with non-transfected myoblasts is unclear.
Taken together, porcine PPAR was able to trigger myoblasts transdifferentiation into adipocytes in the presence of a PPAR-ligand. The current finding is consistent with the hypothesis that decreased phosphorylation of mutated PPAR has enhanced activity on stimulating adipocyte differentiation. Treatment with a PPAR-ligand enhanced the adipogenic effect. Future targeting of the wild-type or mutated PPAR to myoblasts in vivo may provide a mechanism to enhance marbling in pigs.
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Footnotes
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1 This work was supported by National Science Council in Taiwan. 
2 Visiting professor at National Taiwan University.
3 Corresponding author: sding{at}ntu.edu.tw
Received for publication November 4, 2005.
Accepted for publication May 19, 2006.
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Abstract
INTRODUCTION
