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Experimental Physiology 91.4 pp 747-753
DOI: 10.1113/expphysiol.2006.033662
© The Physiological Society 2006
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Upregulation of gene encoding adipogenic transcriptional factors C/EBP{alpha} and PPAR{gamma}2 in denervated muscle

A. Wagatsuma1

1 Department of Biochemical Sciences, National Institute of Fitness and Sports, 1 Shiromizu, Kanoya, Kagoshima 891-2393, Japan


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Muscle denervation induces fatty degeneration in skeletal muscle. However, the possible mechanism(s) remains to be elucidated. To gain insight into the regulation of this process, this study was designed to characterize the expression pattern of genes encoding transcriptional factors that regulate adipogenesis and the terminal differentiation marker of adipocytes in denervated muscle. Female mice underwent surgery to transect the sciatic nerve, and then the gastrocnemius muscles were harvested 5, 10, 20 or 30 days after surgery. The extent of fatty degeneration was assessed as lipid accumulation by Oil Red O staining. The cellular localization of CCCAT/enhancer-binding protein {alpha} (C/EBP{alpha}) and peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2), which play an important role in the regulation of adipocyte differentiation, was assessed by immunohistochemistry. The mRNA levels were analysed using a real-time polymerase chain reaction. After muscle denervation, most muscle fibres atrophied pathologically, and lipid accumulation was observed in the superficial region of the gastrocnemius muscle, suggesting that fatty degeneration occurs in this model. Both C/EBP{alpha} and PPAR{gamma}2 proteins were observed in the interstitial space of denervated muscle but detected in small amounts in normal muscle. The expression levels of C/EBP{alpha} and PPAR{gamma}2 were significantly upregulated 30 days after muscle denervation. The expression levels of fatty acid binding protein 4 (FABP4), which reflects fatty acid metabolism, were decreased slightly at 5 and 10 days and then returned to control levels 30 days after muscle denervation. These findings suggest that muscle denervation-induced fatty degeneration may be mediated through C/EBP{alpha} and PPAR{gamma}2.

(Received 15 February 2006; accepted after revision 7 April 2006; first published online 21 April 2006)
Corresponding author A. Wagatsuma: Department of Biochemical Sciences, National Institute of Fitness and Sports, 1 Shiromizu, Kanoya, Kagoshima 891-2393, Japan. Email: waga{at}nifs-k.ac.jp


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Muscle denervation leads to rapid atrophy (Banker & Engel, 2004) and induces progressive fatty degeneration (Dulor et al. 1998). A similar observation is seen in Duchenne muscular dystrophy (Banker & Engel, 2004) and in transgenic mice with an overexpressed disintegrin and metalloproteinase (ADAM 12) (Kawaguchi et al. 2002) and activated I{kappa}B kinase ß/NF{kappa}B (Cai et al. 2004). It has been demonstrated that muscle denervation upregulates several genes, including leptin, lipoprotein lipase and glycerol 3-phosphate dehydrogenase, associated with adipose tissue (Dulor et al. 1998), suggesting that muscle denervation activates the adipogenic potential in skeletal muscle. Although the exact mechanism(s) initiating fatty degeneration in these pathologies remains to be elucidated (Kawaguchi et al. 2002), specific adipogenic transcriptional factors have been implicated.

In vitro models of adipogenesis have revealed that the differentiation programme appears to be distinctly controlled through the co-ordinated regulation of transcription factors, including members of the CCAAT/enhancer-binding proteins (C/EBP) and peroxisome proliferator-activated receptor (PPAR) families (Cowherd et al. 1999). The family of C/EBP, including C/EBP{alpha}, ß and {delta}, has been implicated in the induction of adipocyte differentiation (Mandrup & Lane, 1997). C/EBPß and C/EBP{delta} induce the expression of C/EBP{alpha} and PPAR{gamma} (Rosen, 2005), while C/EBP{alpha} binds and transactivates the promoters of several adipocyte genes (Gregoire et al. 1998). The gene encoding PPAR{gamma} gives rise to two isoforms, PPAR{gamma}1 and PPAR{gamma}2 (Zhu et al. 1995). PPAR{gamma}2 appears to be more adipose specific than PPAR{gamma}1 (Vidal-Puig et al. 1996) and mediates gene expression related to fatty acid metabolism (Morrison & Farmer, 1999). Therefore, C/EBP{alpha} and PPAR{gamma}2 are critical transcriptional factors in adipocyte differentiation (Rosen et al. 2002). To our knowledge, their pattern of expression is not fully characterized in denervated muscle. Therefore, we hypothesized that the expression of PPAR{gamma}2 and C/EBP{alpha} would be modulated in response to decreased neuromuscular activity, induced by muscle denervation, leading to fatty degeneration.

To test this hypothesis, this sudy was designed to characterize the expression pattern of genes encoding transcriptional factors that regulate adipogenesis and the terminal differentiation marker of adipocytes in denervated muscle. Here, it is demonstrated that the expression levels of C/EBP{alpha} and PPAR{gamma}2 were upregulated concomitant with lipid accumulation in denervated muscle. This finding suggests that muscle denervation-induced fatty degeneration may be mediated through C/EBP{alpha} and PPAR{gamma}2.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animal care and surgical procedure

Female 7-week-old CD1 mice (CLEA, Tokyo, Japan) were used in the present study. The mice were randomly assigned to two groups, the first group (n = 36) had the gastrocnemius muscle denervated, while the second group (n = 36) was not denervated and served as a control group. The mice were housed in the animal care facility under a 12 h light–12 h dark cycle at room temperature (23 ± 2°C) and 55 ± 5% humidity. The mice were maintained on a diet of CE-2 rodent chow (CLEA) and given water ad libitum. All procedures in the animal experiments were performed in accordance with the guidelines presented in the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences, published by the Physiological Society of Japan. This study was also approved by the Animal Committee of the National Institute of Fitness and Sports, Japan.

A surgical level of anaesthesia was induced by an intraperitoneal injection of sodium pentobarbitone (30 mg kg–1). All surgical procedures were performed under aseptic conditions. The right sciatic nerve was isolated mid-thigh and transected 5–8 mm proximal to the trifurcation. The mice were killed by cervical dislocation. Gastrocnemius muscles (superficial portion) were harvested 30 days after surgery and immediately stored for histochemical and biochemical analysis.

Histochemistry and immunohistochemistry

The gastrocnemius muscle was dissected from mice and frozen in liquid nitrogen-cooled isopentane. Transverse sections (8–10 µm thickness) were cut using a cryostat at –20°C and thawed on 3-amino propylethoxysilane-coated slides. Lipid accumulation in muscle tissue was assessed by Oil Red O staining (Koopman et al. 2001). Briefly, the sections were fixed with 3.7% formaldehyde for 60 min and rinsed with deionized water. The sections were incubated with Oil Red O solution for 30 min and rinsed with deionized water. The nuclei were counterstained with Haematoxylin, and the sections were mounted with 10% glycerol in phosphate-buffered saline (PBS). Morphometric measurements were made using light microscopy and image collection with a CCD camera. Fibre cross-sectional area (FCSA) and lipid accumulation area were measured in selected fields (size of analysed field, 310 µm x 410 µm) in the superficial region of the gastrocnemius muscle. Fibre cross-sectional area was measured in at least 100 muscle fibres per sample. Lipid accumulation area was measured in two-to-three analysed fields per sample and expressed as the area per millimetre sqared of muscle section.

For immunohistochemical analysis, the sections were fixed with cold acetone (–20°C) for 10 min and then washed with 0.1 M PBS. To quench endogenous peroxidase activity, the sections were incubated with a solution of methanol containing 1% H2O2 for 60 min. The sections were washed with 0.1 M PBS, blocked with 0.1 M PBS containing 1% bovine serum albumin and 0.1% Triton X-100, and then incubated overnight at 4°C with rabbit anti-C/EBP{alpha} antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-PPAR{gamma}2 (1:50; Alexis Biochemicals, San Diego, CA, USA) in 0.1 M PBS containing 1% bovine serum albumin and 0.1% Triton X-100. The sections were incubated with horseradish peroxidase-conjugated secondary antibody (1:500; Molecular Probes, Eugene OR, USA) in 0.1 M PBS containing 1% bovine serum albumin and 0.1% Triton X-100 for 30 min at room temperature. To detect immunoreactive protein, the sections were incubated with 0.1 M PBS containing 0.05% 3-diaminobenzidine and 0.01% H2O2 for 10 min at room temperature.

RNA extraction and cDNA synthesis

The gastrocnemius muscles were carefully harvested, and the tissue was then transferred to glass homogenizers on ice, and 1 ml TRI reagent (Molecular Research Center, Cincinnati OH, USA) was added per 10–20 mg tissue. RNA integrity was confirmed by denaturing agarose gel electrophoresis, and the concentration was quantified by measuring the optical density (OD) at 260 nm. All samples had an optical density ratio (OD260/OD280) of at least 1.9. The DNase-treated total RNA (1 µg) was then converted to cDNA using a First-strand cDNA synthesis system for quantitative real-time polymerase chain reaction (Marligen Biosciences, Ijamsville, MD, USA). The cDNA samples were aliquoted and stored at –80°C.

Real-time polymerase chain reaction (RT-PCR) analysis

Real-time PCR was performed using an OpticonTM DNA Engine (MJ Research, Waltham MA, USA) according to the manufacturer's instructions. Amplification was carried out using SYBR Premix Ex TaqTM (TaKaRa Bio, Shiga, Japan). All primers used in this study were obtained from Espec Oligo Service (Ibaraki, Japan). The reactions employed primers for MyoD, myogenin (Warren et al. 2002), CCAAT/enhancer binding protein (C/EBP{alpha}), peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2; Tanabe et al. 2004), fatty acid binding protein 4 (FABP4, previously known as aP2; Taylor-Jones et al. 2002), and ß-actin (Real Time PCR Primer and Probe Database; http://medgen.ugent.be/rtprimerdb/). For each set of primers, PCR thermal cycle conditions were optimized to achieve a single ethidium bromide-stained band following electrophoresis on a 2% agarose gel. Differences in gene expression were calculated relative to the expression of ß-actin and compared to a standard curve. ß-Actin was confirmed to be appropriate for normalizing the signal by comparing the differences in raw threshold cycle values (the number of amplification cycles at which the signal is detected above the background and is in the exponential phase). A standard curve was constructed from serially diluted cDNA from gastrocnemius muscle. Each sample was normalized to its ß-actin content. The final results were expressed as a relative fold-change compared to control animals.

Statistical analysis

Data are expressed as means ± S.E.M. The data were compared using unpaired Student's t test. The real-time PCR data were compared using one-way analysis of variance (ANOVA) with Fisher's least significant difference (LSD) test. A 0.05 level of probability was used as the criterion for statistical significance.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
After muscle denervation, FCSA was significantly decreased by 76.8% relative to control muscle (Fig. 1). As shown in Fig. 2, control muscle fibres were characterized by regular outlines and peripherally located nuclei, whereas denervated muscle fibres were pathologically atrophied. To assess fatty degeneration, the cryostat sections were stained with Oil Red O. No apparent lipid accumulation was observed in control muscle, while in denervated muscle lipid accumulation was restricted to the superficial region of gastrocnemius muscle and frequently found near blood vessels. Figure 3 shows quantitative analysis of lipid accumulation in skeletal muscle. To determine the cellular localization of C/EBP{alpha} and PPAR{gamma}2, we performed immunohistochemical analysis in normal and denervated muscles. Both C/EBP{alpha} and PPAR{gamma}2 proteins were observed in the interstitial space of denervated muscle but also detected in small amounts in normal muscle (Fig. 4).


Figure 1
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  		Figure 1.  Effect of muscle denervation on fibre cross-sectional area (FCSA)
Data are expressed as means ± S.E.M. (control, n = 6; muscle denervation, n = 6) ***P < 0.001 compared with control muscle

 

Figure 2
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  		Figure 2.  Effects of muscle denervation on lipid accumulation in control (A) and denervated muscles (B and C)
No lipid accumulation was observed in the control muscle (A). Lipid accumulation is apparent in denervated muscle (B) and is frequently found in the perivascular space (C). Scale bar represents 50 µm

 

Figure 3
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  		Figure 3.  Quantification of the areas stained with Oil Red O on a cryostat section
Data are expressed as means ± S.E.M. (control, n = 6; muscle denervation, n = 6). ***P < 0.001 compared with control muscle

 

Figure 4
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  		Figure 4.  Immunohistochemical analysis of C/EBP{alpha} and PPAR{gamma}2 in skeletal muscle
Spatial distribution of C/EBP{alpha} protein in control (A) and 30 days denervated muscle (B). Spatial distribution of PPAR{gamma}2 protein in control (C) and 30 days denervated muscles (D). Immunoreactivity is indicated by arrows. (control, n = 3; muscle denervation, n = 3). Scale bar represents 50 µm

 
To characterize the expression pattern of myogenic, adipogenic transcriptional factors and terminal differentiation marker of adipocytes in denervated muscle, we investigated mRNA levels using a real-time PCR. MyoD, myogenin, C/EBP{alpha}, PPAR{gamma}2 and FABP4 mRNA transcripts were readily detected in control and denervated muscles. As shown in Fig. 5, the expression levels of MyoD and myogenin were significantly increased in the denervated muscle relative to control muscle. The expression levels of C/EBP{alpha} and PPAR{gamma}2 were significantly upregulated 30 days after muscle denervation. The expression levels of fatty acid binding protein 4 (FABP4) was slightly reduced at 5 and 10 days and then returned to control levels 30 days after muscle denervation.


Figure 5
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  		Figure 5.  Effects of muscle denervation on expression levels of myogenic and adipogenic transcriptional factors and terminal differentiation marker of adipocytes
The amount of target gene expression was normalized to the expression of the ß-actin gene. Experiments were performed in duplicate, and data are expressed as means ± S.E.M. (control, n = 6–12 for each time point; muscle denervation, n = 6–12 for each time point). *P < 0.05, **P < 0.01 compared with control muscle

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The expression of myogenic transcriptional factors is rapidly and transiently induced in adult skeletal muscle in response to muscle denervation (Eftimie et al. 1991). We observed stable elevated levels of MyoD and myogenin mRNA for 30 days after muscle denervation. This finding is generally consistent with a previous study (Adams et al. 1995), although the implication of reactivation of the myogenic program during muscle denervation is not fully understood. MyoD protein is expressed in satellite cells of denervated muscle (Ishido et al. 2004), suggesting that satellite cells have been activated. Therefore, most of the activated satellite cells can differentiate into myogenic lineage. However, satellite cells are known to be multipotential stem cells that exhibit myogenic, osteogenic and adipogenic differentiation in vitro under appropriate conditions (Asakura et al. 2001). This in vitro study allows us to hypothesize that some satellite cells could differentiate into adipocyte-like cells in response to muscle denervation. Recently, it has been reported that myogenic and adipogenic programmes are coexpressed in myogenic cells from Wnt10b-deficient mice (Vertino et al. 2005). Wnt signalling, probably mediated by Wnt10b, seems to inhibit adipogenic differentiation through inhibition of the adipogenic transcriptional factors C/EBP{alpha} and PPAR{gamma} (Ross et al. 2000). Therefore, Wnt10b may possibly regulate adipogenic potential in satellite cells of denervated muscle.

We observed both C/EBP{alpha} and PPAR{gamma}2 proteins in the interstitial space of denervated muscle, suggesting that most of the cells which activate adipogenic potential, might be derived from preadipocytes and mesenchymal progenitors. This possibility may be partly supported by the observation that the CD34-positive and CD45-negative cells in the interstitial space of skeletal muscle have potential to differentiate into adipocytes cells in vitro (Tamaki et al. 2002). The CD34-positive and CD45-negative cell showed oil droplet-like staining typical of fat cells and was distinct from satellite cells. However, it remains unclear whether the CD34-positive and CD45-negative cell can differentiate into an adipocyte in denervated muscle.

Two adipogenic transcriptional factors, C/EBP{alpha} and/or PPAR{gamma}, have been implicated in the co-ordinated activation of adipocyte-specific genes (Gregoire et al. 1998). C/EBP{alpha} functions as a trans-activator of aP2 gene promoter (Christy et al. 1989). Similarly, PPAR{gamma}2 forms a heterodimeric complex with retinoid X receptor {alpha} (RXR{alpha}), which can bind to and activate the aP2 enhancer (Tontonoz et al. 1994). We observed that C/EBP{alpha} and PPAR{gamma}2 were upregulated in denervated muscle, suggesting that increased expression of adipogenic transcriptional factors potentially contribute to FABP4 gene expression. The expression of FABP4 was reduced at 5–10 days and returned to control levels 30 days after muscle denervation concomitant with increased expression of C/EBP{alpha} and PPAR{gamma}2. Therefore, it is possible that it takes a longer time to accumulate FABP mRNA after C/EBP{alpha} and PPAR{gamma}2 are activated in denervated muscle. Intriguingly, when myoblasts from adult mice are cultured under adipocyte-inducing medium, C/EBP{alpha} and PPAR{gamma}2 mRNAs accumulate but aP2 mRNA does not (Taylor-Jones et al. 2002), suggesting that the activities of transcriptional factors can be regulated post-transcriptionally. For example, mitogen-activated protein kinase-mediated phosphorylation of PPAR{gamma} (Hu et al. 1996) and RXR{alpha} (Solomon et al. 1999) reduces their activities, while protein kinase A-mediated phosphorylation of PPAR{gamma} enhances its activity (Lazennec et al. 2000). Furthermore, aP2 mRNA accumulates in cultured myoblasts from aged mice concomitant with decreased expression of Wnt10b mRNA (Taylor-Jones et al. 2002), suggesting that Wnt10b may play a role in expression of aP2, probably through C/EBP{alpha} and PPAR{gamma}2. Therefore, further studies are necessary to clarify the role of these transcriptional factors in fatty degeneration induced by muscle denervation.

Consistent with a previous study (Vertino et al. 2005), we observed no apparent lipid accumulation in control muscle, suggesting that activation of adipogenic potential may be inhibited under physiological conditions. The lipid accumulation observed in the denervated muscle suggests that adipogenic potential has been activated. Additionally, lipid accumulation was restricted to the superficial region of the gastrocnemius muscle, which is composed of predominantly fast-twitch muscle fibres. This finding is in agreement with a previous study showing that the kinetics of fatty degeneration is different in fast-twitch and slow-twitch muscles, the former being transformed faster and more completely than the latter (Dulor et al. 1998).

The observed lipid accumulation near blood vessels is consistent with the observations in transgenic mice with an overexpressed ADAM12 (Kawaguchi et al. 2002). This suggests the possibility that the adipocytes originate from mesenchymal progenitor/preadipocytes in the perivascular space. However, the origin of the cells that accumulate lipid remains unknown. In our model, myogenic and adipogenic transcriptional factors were upregulated in denervated muscle. This observation leads us to speculate that the number of cells activating both adipogenic and myogenic potentials may be low because C/EBP{alpha} and PPAR{gamma}2 have been shown to inhibit myogenic differentiation by downregulating the MyoD family genes in myogenic cells (Hu et al. 1995). Although we did not identify the kind of cell that expresses adipogenoic transcriptional factors, we speculated that preadipocytes would proliferate and differentiate into adipocytes in response to muscle denervation. This possibility may be supported by the observation that sympathetic denervation of white adipose tissue induces preadipocyte proliferation and increases the number of adipocytes (Cousin et al. 1993), suggesting that muscle denervation may stimulate preadipocyte proliferation and accelerate adipocyte differentiation. However, we cannot rule out the possibility that satellite cells (Asakura et al. 2001), and a side population of skeletal muscle-derived stem cells (Jackson et al. 1999), could also differentiate into adipocyte-like cells during muscle denervation; their contribution to fatty degeneration is currently unknown.

In conclusion, we have provided the first evidence that two adipogenic transcriptional factors, C/EBP{alpha} and PPAR{gamma}2, were upregulated in denervated muscle where lipid accumulation was observed. Although the mechanism by which muscle denervation regulates these adipogenic transcriptional factors remains to be identified, these findings may explain, at least in part, why lipids accumulate in denervated muscle.


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 Introduction
 Methods
 Results
 Discussion
 References
 
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   Acknowledgements
 
We would like to thank Kelly McGrath for a critical reading of the manuscript. This study was supported, in part, by the Grant-in-Aid for Scientific Research (B-17700499 to A. Wagatsuma) from the Japan Society for the Promotion of Science, and the Grant-in-Aid for Scientific Research from the National Institute of Fitness and Sports of Japan (President's Discretionary Budget, 2005, to A. Wagatsuma).





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