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Increased adipogenicity of cells from regenerating skeletal muscle
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
Increased adipogenicity of cells from regenerating skeletal muscle Keitaro Yamanouchi ⁎, Erica Yada, Naomi Ishiguro, Tohru Hosoyama, Masugi Nishihara Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
ARTICLE INFORMATION
ABS T R AC T
Article Chronology:
Adipose tissue development is observed in some muscle pathologies, however,
Received 24 December 2005
mechanisms that induce accumulation of this tissue as well as its cellular origin are
Revised version received
unknown. The adipogenicity of cells from bupivacaine hydrochloride (BPVC)-treated and
4 April 2006
untreated muscle was compared in vitro. Culturing cells from both BPVC-treated and
Accepted 6 April 2006
untreated muscles in adipogenic differentiation medium (ADM) for 10 days resulted in the
Available online 5 June 2006
appearance of mature adipocytes, but their number was 3.5-fold higher in cells from BPVCtreated muscle. Temporal expressions of PPARγ and the presence of lipid droplets during
Keywords:
adipogenic differentiation were examined. On day 2 of culture in ADM, only cells from BPVC-
Skeletal muscle
treated muscle were positive both for PPARγ and lipid droplets. Pref-1 was expressed in cells
Adipocytes
from untreated muscle, whereas its expression was absent in cells from BPVC-treated
Adipose tissue development
muscle. In ADM, the presence of insulin, which negates an inhibitory effect of Pref-1 on
Adipogenesis
adipogenic differentiation, was required for PPARγ2 expression in cells from untreated
Progenitor cells
muscle, but not for cells from BPVC-treated muscle. These results indicate that BPVCinduced degenerative/regenerative changes in muscle lead to increased adipogenicity of cells, and suggest that this increased adipogenicity not only involves an increase in the number of cells having adipogenic potential, but also contributes to the progression of these cells toward adipogenic differentiation. © 2006 Elsevier Inc. All rights reserved.
Introduction In some pathological conditions, increased adipose tissue development (accumulation of adipocytes) is observed in skeletal muscle [1,2]. Adipose tissue development in skeletal muscle is widely recognized as one of the hallmarks of pathological changes seen in Duchenne muscular dystrophy (DMD) [3,4]. It has been described that in advanced cases of DMD, repeated degeneration and regeneration of skeletal muscle fibers finally result in loss of muscle fibers, and this is correlated with an accumulation
of adipocytes in the area where muscle fibers are lost [5]. In addition, the accumulation of adipocytes in skeletal muscle often coincides with sarcopenia (loss of skeletal muscle mass due to atrophy of muscle fibers) [6]. Although mechanisms that induce the accumulation of adipocytes, as well as the cellular origins of adipocytes in skeletal muscle, are unclear, the fact that adipose tissue development is associated with degenerative/regenerative or atrophic changes in skeletal muscle fibers suggests that these changes in skeletal muscle fibers could lead to increased adipogenicity of the cells in skeletal muscle.
⁎ Corresponding author. Fax: +81 3 5841 8017. E-mail address: [email protected] (K. Yamanouchi). 0014-4827/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2006.04.014
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Adipogenic differentiation is under the control of several genes such as preadipocyte factor-1 (Pref-1) [7], peroxisome proliferator activating receptor-γ (PPARγ) [8–11] and CCAAT/ enhancer binding protein-α (C/EBPα) [12]. Pref-1 is a member of the epidermal growth factor (EGF)-like protein family and is abundantly expressed in preadipocytes, whereas its expression in differentiating adipocytes is undetectable [7]. Forced expression of Pref-1 was reported to inhibit adipogenesis; accordingly, Pref-1 was suggested to play an important role in the maintenance of the undifferentiated preadipose state [7]. Terminal differentiation of preadipocytes to adipocytes is believed to be under the control of PPARγ and C/EBPα [13–15]. It has been accepted that once activated, PPARγ and C/EBPα cross-regulate each other to maintain their gene expressions [16,17], and both alone or in cooperation with each other can induce terminal differentiation of adipocytes [9,10,18,19]. Skeletal muscle contains several types of progenitor cells including satellite cells that reside beneath the basal lamina of muscle fiber [20] and CD34+/CD45− cells (SK34 cells) that reside in interstitial spaces of skeletal muscle [21]. Among these cells, recent observation indicates that satellite cells possess the capability to be myogenic stem cells, as well as multipotent stem cells, since satellite cells being cultured in the presence of specific cell lineage inducing cocktails can differentiate into several types of mesenchymal lineages, such as osteocytes and adipocytes [22,23]. In addition, Sk34 cells can be divided into two populations, namely side population (SP) cells and main population (MP) cells, based on the efflux of fluorescent dye Hoechst 33342. Both SP-Sk34 and MP-Sk34 cells also possess adipogenic potential [21]. Bupivacaine hydrochloride (BPVC), a local anesthetic, is widely used for studies on skeletal muscle regeneration. It is known to induce widespread degeneration and necrosis, followed by regeneration of skeletal muscle fibers, without affecting satellite cell viability [24]. A number of studies have been carried out on the BPVC-induced model of skeletal muscle degeneration/regeneration [25–27]. We hypothesized that degenerative/regenerative changes in skeletal muscle could lead to increased adipogenicity of cells in skeletal muscle. Therefore, we have employed a BPVCinduced model of skeletal muscle degeneration/regeneration, and compared the adipogenicity of cells derived from BPVCtreated muscle to those from untreated muscle by culturing them in adipogenic differentiation medium.
Materials and methods Animals and skeletal muscle regeneration Adult male rats (6 to 9 months) of the Wistar Imamichi strain were used throughout this study. They were bred in our laboratory and housed in a room at 23°C with 14 h of light and 10 h of darkness (lights on at 0500 h). Food and water were provided ad libitum. Under light ether anesthesia, 100 μl of bupivacaine hydrochloride (BPVC; 0.75% in saline) was injected into the tibialis anterior (TA) muscles of both legs. Two days after BPVC injection, they were used for isolation of mononucleated cells as described below. As a control, mononucleated cells were
obtained from a rat without BPVC treatment, since BPVC injection into the TA muscle of one side might affect the cells in the TA muscle of the other side. All animal experiments performed in this study were according to the Guideline for the Care and Use of Laboratory Animals, The University of Tokyo.
Isolation and culture of mononucleated cells from skeletal muscle Procedures, previously described by Allen et al. [28] for isolating skeletal muscle satellite cells, were applied to isolate mononucleated cells from skeletal muscle. Briefly, rats injected with BPVC (BPVC-treated) or not injected (control) were sacrificed by inhalation of carbon dioxide gas. The TA muscles were excised, trimmed of fat and connective tissue, hand-minced with scissors and digested for 1 h at 37°C with 1.25 mg/ml pronase (protease; Sigma, MO). Cells were separated from muscle fiber fragments and tissue debris by differential centrifugation and plated on poly-L-lysine and fibronectin-coated dishes in Dulbeccos' modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 50 U/ml penicillin, 50 μg/ml streptomycin and 50 μg/ml gentamicin (10%FBS/DMEM). Typically, a 24-well culture plate was used, and cells were plated at a density based on the starting tissue weight when the cells were isolated (0.5 g of muscle tissues per one well/500 μl 10% FBS/DMEM). Since BPVC treatment is known to increase the cell yield per gram tissue weight [29], thus the initial number of cells plated differed between BPVCtreated and control rats. Cultures were maintained in a humidified atmosphere of 5% CO2 at 37°C. For experiments in which cells were cultured in adipogenic differentiation medium, freshly isolated cells were initially plated in 10% FBS/DMEM and cultured for one day to ensure cell attachment onto the culture surface. Medium was replaced with adipogenic differentiation medium one day after plating (designated as day 0). Adipogenic differentiation medium consisted of the following: from day 0 to day 2, 10% FBS/DMEM supplemented with insulin (1 μg/ml), dexamethazone (0.1 μg/ ml), isobutylmethylxanthine (IBMX; 27.8 μg/ml ) and troglitazone (10 μM; kindly donated by Sankyo Co. Ltd.), from day 2 to day 4, 10% FBS/DMEM supplemented with insulin and troglitazone, and thereafter, 10% FBS/DMEM supplemented only with troglitazone. Medium was replaced every other day until the days indicated in the results. Cells not induced to adipogenically differentiate were cultured in 10% FBS/DMEM only. Additionally, experiment to examine if the difference in the initial cell density between BPVC-treated and control rats could affect the overall results in the present study was performed. In this experiment, cells isolated from BPVC-treated and control TA muscles were plated at same density (6.7 × 105 cells/cm2). One day after plating, cells were fed with adipogenic differentiation medium and cultured for 2 days. The cells were then immunocytochemically stained with anti-PPARγ (described below).
Immunocytochemistry Single staining of MyoD, PPARγ and C/EBPα Unless otherwise stated, all procedures were performed at room temperature (RT). Cultured cells were fixed with 4% paraformaldehyde (PFA) in phosphate buffered-saline (PBS) for 15 min
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(for MyoD and PPARγ) or with ice-cold methanol at − 20°C (for C/ EBPα) for 10 min. Cells were washed with PBS and incubated for 15 min in 5% normal goat serum (NGS) and 0.6% hydrogen peroxide in PBS (blocking solution) to block non-specific binding of antibodies and to quench endogenous peroxidase activity. For MyoD and PPARγ staining, 0.1% triton X-100 was also included in the blocking solution. After washing with PBS, mouse anti-MyoD (1:100 diluted with 5% NGS in PBS; clone 5.8A, Novocastra, Newcastle upon Tyne, UK), anti-PPARγ (1:100 diluted with 5% NGS in PBS; E-8, sc-7273, Santa Cruz, CA) or rabbit anti-C/EBPα antibody (1:200 diluted with 5% NGS in PBS; 14AA, sc-61, Santa Cruz, CA) was applied, and the cells were incubated for 3 h. After washing cells with PBS again, horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Simple stain rat MAX-PO for rabbit IgG; Nichirei, Tokyo, Japan) or HRP-conjugated anti-mouse IgG (Simple stain rat MAX-PO for mouse IgG; Nichirei) was added and incubated for 1 h. Cells were then washed with PBS, and signals were visualized by incubating cells in diaminobenzidine (0.5 mg/ml) and hydrogen peroxide (0.03%) in PBS. In some experiments, cell nuclei were counterstained with hematoxylin and/or cells were doublestained with Oil Red-O (described below) after immunostaining. When quantitative analyses on immunopositive cells and lipid droplet-positive cells were performed, total number of cells and number of positive cells obtained from 10 different fields randomly chosen microscopically using a 40× objective were counted. Cell numbers and the percentages of positive cells were then averaged for the triplicate culture wells. Typically, 150 to 800 cells were counted per well.
Single staining of Pref-1 All procedures were performed at RT. Cultured cells were fixed with 4% PFA in PBS for 15 min, washed with PBS, and incubated for 15 min in 5% NGS in PBS (blocking solution) to block non-specific binding of the antibody. After washing with PBS, rabbit anti-Pref-1 (anti-DLK) antibody (1:100 diluted with 5% NGS in PBS; H-118, sc-25437, Santa Cruz) was applied, and cells were incubated for 3 h. After washing with PBS again, cells were incubated for 1 h in AlexaFluor 594 conjugated-antirabbit IgG (1:200 diluted with 5% NGS in PBS; Invitrogen, CA). Cells were washed with PBS, and nuclei were counterstained with Hoechst 33258. Observations were made using a fluorescence microscope equipped with a digital camera (DP70, Olympus, Tokyo, Japan).
Double staining of C/EBPα and ED1 Unless otherwise stated, all procedures were performed at RT. Cultured cells were fixed with ice-cold methanol at −20°C for 10 min, washed with PBS, and incubated for 15 min in 5% NGS in PBS (blocking solution) to block non-specific binding of the antibodies. After washing with PBS, a mixture of rabbit anti-C/ EBPα antibody (1:200 diluted with 5% NGS in PBS) and mouse anti-ED1 (1:400 diluted with 5% NGS in PBS; BMA Biomedicals AG, Rheinstrasse, Switzerland) was applied, and cells were incubated for 3 h. After washing with PBS again, cells were incubated for 1 h in a mixture of AlexaFluor 488 conjugatedanti-rabbit IgG (1:400 diluted with 5% NGS in PBS; Invitrogen) and AlexaFluor 594 conjugated-anti-mouse IgG (1:400 diluted with 5% NGS in PBS; Invitrogen). Cells were washed with PBS, and nuclei were counterstained with Hoechst 33258. Observa-
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tions were made using a fluorescence microscope equipped with a digital camera.
Oil Red-O staining Cultured cells were fixed with 4% PFA in PBS for 10 min, washed with PBS and stained in Oil Red-O solution (2:3 mixture of 0.5% (w/v) Oil Red-O in 2-isopropanol and distilled water) for 8 min, then washed with PBS. In some experiments, quantitative analyses of Oil Red-O stained cells were performed. Ten days after culture, when cells were at 100% confluency, six different fields randomly chosen under the microscope using a 20× objective were photographed, and areas occupied with Oil RedO-positive cells were quantified using NIH Image software (ver. 1.62, NIH). Values representing incidence of adipogenesis (where applicable) were expressed as the mean pixel measurements for the triplicate culture wells.
RT-PCR Total RNA was isolated from cultured cells at the indicated time points using Trizol reagent. The cDNA was synthesized from 2 μg of total RNA by SuperScript II (Invitrogen) using oligo-dT primer. The PCR was performed on mRNA transcripts of ap2, Pref-1, PPARγ2 and hypoxanthine guanine phosphoribosyltransferase (HPRT), using αTaq polymerase (Bionex, Seoul, Korea). Primer sets for ap2, Pref-1, PPARγ2 and HPRT, annealing temperatures, as well as cycle numbers for corresponding primer sets are as follows: For ap2: Forward 5′-AGCTTGTCTCCAGTGAAAAC-3′, Reverse 5′-GAAGTCACGCCTTTCATAAC-3′, annealing temperature 55°C, 32 cycles. For Pref-1: Forward 5′-GGAAGGCTGGGACGGGAAAT-3′, Reverse 5′- GACACTCGAAGCTCACCTGG-3′, annealing temperature 58°C, 32 cycles. For PPARγ2: Forward 5′-TTCGCTGATGCACTGCCTAT-3′, Reverse 5′-GCCAACAGCTTCTCCTTCTC-3′, annealing temperature 58°C, 30 cycles. For HPRT: Forward 5′-GCTGGTGAAAAGGACCTCT-3′, Reverse 5′-CACAGGACTAGAACRYCTGC-3′, annealing temperature 58°C, 29 cycles (R stands for A or G; Y stands for C or T; these were included so that this primer set could be used both for rat and mouse samples in our laboratory). Annealing temperatures and cycle numbers were determined so that the amplification reactions were within the linear range. The PCR products were visualized with ethidium bromide after agarose-gel electrophoresis. Quantitative analysis on the mRNA expression levels was performed on the photo of the gel using NIH image software, and the data are indicated as pixel.
Statistical analyses Each experiment was repeated two to three times, and only representative data are shown. The data are expressed as means ± SE. Statistical analyses were performed using commercial software (StatView, ver. 4.5, Abacus Concepts, Inc., Berkley, CA). Student's t test or one-way ANOVA followed by Bonferroni's test were used to evaluate statistical
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differences among the groups. P values less than 0.05 were considered as statistically significant.
Results BPVC-treatment increases the number of cells capable of differentiating into mature adipocytes In order to determine if BPVC-treatment increases the total number of cells capable of differentiating into mature adipocytes, mononucleated cells were isolated from control and BPVC-treated TA muscles and cultured with or without adipogenic stimulation for 10 days. After 10 days of culture, when cells under any condition were nearly 100% confluent, Oil Red-O staining was used to determine the degree to which cells had differentiated into mature adipocytes . In the absence of adipogenic stimulation, no cells were stained with Oil Red-O. On the other hand, in the presence of adipogenic stimulation, some cells from control TA muscle acquired a mature adipocyte-like phenotype, as represented by Oil Red-O staining, while the incidence of adipogenesis was significantly increased (>3.5-fold; P < 0.05) in cells from BPVC-treated TA muscle (Fig. 1). In addition, mRNA expression levels of ap2, a marker representing for the presence of mature adipocytes, were compared between the cells from BPVC-treated and control TA muscles cultured with adipogenic differentiation medium for 4 days. As shown in Fig. 2, more lipid droplet-positive cells were seen in the cells from BPVC-treated TA muscle and accordingly, mRNA expression level of ap2 was also increased (>2-fold; P < 0.01) in the cells from BPVC-treated TA muscle compared to those from control TA muscle. These results indicate that in response to BPVC-treatment, the number of cells capable of differentiating into mature adipocytes increases.
Expressions of MyoD, PPARγ and C/EBPα in cells from control and BPVC-treated TA muscles Mononucleated cells were prepared from BPVC-treated or control TA muscle and their expressions of MyoD, PPARγ and
C/EBPα were examined on day 2 after plating in 10% FBS/ DMEM. Nearly all cells (>95%) from control TA muscle exhibited positive MyoD staining, whereas only a portion of cells from BPVC-treated TA muscle were positive for MyoD (Fig. 3A), indicating the presence of non-myogenic cells. This was in agreement with the previous result shown by Molnar et al. [29]. PPARγ and C/EBPα were negligible in cells from control TA muscle (Fig. 3B and C), indicating the absence of differentiated adipocytes in control TA muscle. Cells from BPVC-treated TA muscle were also negative for PPARγ (Fig. 3B), but rich in C/EBPα-positive cells (Fig. 3C). However, a vast majority of C/EBPα-positive cells were either co-stained with anti-ED1, a marker for invading macrophages [30] (Fig. 3C) or anti-ED2, a marker for resident macrophages [30] (data not shown). Thus, these results indicate that without adipogenic stimulation, there are no differentiated adipocytes in cells from BPVCtreated TA muscle.
Rapid differentiation into adipocytes of cells from BPVC-treated TA muscle upon adipogenic stimulation Although there were no differentiated adipocytes in cells from BPVC-treated TA muscle, the results shown in Fig. 1 and 2 indicated increased adipogenicity of cells from BPVC-treated TA muscle upon adipogenic stimulation. In order to know whether this is due to the mere increase in number of cells that are capable of differentiating into adipogenic lineage in response to BPVC treatment, and/or is attributed to the appearance of cells that are proceeding to adipogenic differentiation, cells from control and BPVC-treated TA muscle were cultured in adipogenic differentiation medium for 2 days, and PPARγ, C/EBPα expressions and the presence of lipid droplets were examined. As expected and shown in Fig. 4A, number of cells from BPVC-treated TA muscle on day 2 of culture was 2.4fold higher (p<0.01) than that from control TA muscle. No cells positive for lipid droplets/PPARγ were seen in the culture from control TA muscle (Fig. 4B). On the other hand, 7.9% of the cells from BPVC-treated TA muscle were double-positive for PPARγ and lipid droplets (Fig. 4B). Similar result was obtained when the initial cell density was matched between the cells form
Fig. 1 – Appearance of Oil Red-O positive cells in response to adipogenic stimulation in vitro. Mononucleated cells from BPVC-treated or untreated (control) TA muscles were cultured with or without adipogenic stimulation for 10 days. Graphed data indicates the quantitative analysis on the number of Oil Red-O positive cells. Data are expressed as mean ± SE (n = 3).
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Fig. 2 – RT-PCR analysis on ap2 mRNA expression levels in the cells from BPVC-treated and control TA muscles. Cells were cultured with adipogenic stimulation for 4 days. (A) Lipid droplet-positive cells were indicated by white arrowheads. (B) Quantitative analysis of ap2 mRNA expression levels. The data are expressed as means ± SE (n = 3). There was no statistical difference in HPRT mRNA expression levels. neg, negative control (PCR without template).
Fig. 3 – Immunocytochemical analyses of myogenic and adipogenic markers in mononucleated cells from BPVC-treated or untreated (control) TA muscles. Cells were cultured in 10% FBS/DMEM for 2 days. (A) MyoD. Black and white arrowheads indicate positive and negative cells, respectively. (B) PPARγ. No positive cells were seen. (C) C/EBPα. Cells were also double-stained for C/EBPα and ED1. Note that most C/EBPα-stained cells are positive for ED1. Black arrowheads in panel C (middle) indicate C/EBPα-positive cells. Black and white arrowheads (C, right) indicate C/EBPα-positive but ED1-negative cells and C/EBPα/ED1 double-positive cells, respectively.
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Fig. 4 – Quantitative analyses of adipogenic differentiation of cells from BPVC-treated and untreated (control) TA muscle on day 2 of culture with adipogenic stimulation. Cells were cultured in adipogenic differentiation medium for 2 days. (A) Total number of cells. (B) Percentages of lipid droplet/ PPARγ double-positive cells. (C) C/EBPα-immunostaining and Oil Red-O staining. Black and white arrowheads indicate C/EBPα/Oil Red-O double-positive cell and C/EBPα-positive but Oil Red-O-negative cell, respectively. Graphed data (A and B) are expressed as mean ± SE (n = 3).
BPVC-treated and control TA muscles (PPARγ-positive cells, 1.8 ± 0.9% (control, n = 3) vs. 21.7 ± 0.9% (BPVC-treated, n = 3)). C/ EBPα-positive cells were present in both cultures (Fig. 4C), but the percentage of C/EBPα-positive cells was much higher in cells from BPVC-treated TA muscle (26.9%) than in cells from control TA muscle (1.5%). This might be due to the presence of macrophages present in the culture from BPVC-treated TA muscle as was shown in Fig. 3C. However, among the cells from BPVC-treated TA muscle that are positive for C/EBPα, some cells co-stained with Oil Red-O, suggesting the presence of mature adipocytes. Taken together, these results suggest that increased adipogenicity of cells from BPVC-treated TA muscle involves progression of these cells toward adipogenic differentiation, though without adipogenic stimulation, they are neither expressing PPARγ nor C/EBPα as yet.
Time course of PPARγ and C/EBPα expression in cells from BPVC-treated TA muscle with or without adipogenic stimulation To examine thoroughly expressions of PPARγ and C/EBPα during adipogenic differentiation of cells from BPVC-treated TA muscle, time course studies were carried out. For PPARγ expression, no cells were positive on day 0 of culture (Fig. 5A and B). PPARγpositive cells appeared on day 2 of culture when the cells were cultured in adipogenic differentiation medium, confirming the result shown in Fig. 4B, and the percentage of PPARγ-positive
cells was maintained on day 4 of culture (Fig. 5A and B). On the other hand, no PPARγ-positive cells were observed up to 4 days when they were cultured in 10% FBS/DMEM (Fig. 5B). Since PPARγ2 is known to be a highly specific marker of adipocytes [11], its mRNA expression was determined by means of RT-PCR. In agreement with the result obtained by PPARγ-immunocytochemistry, the expression level of PPARγ2 mRNA was negligible on day 0 of culture, but it became apparent on days 2 and 4 of culture in response to adipogenic stimulation (Fig. 5C). C/EBPα-positive cells were observed at all time points examined regardless of adipogenic stimulation (Fig. 6A). However, C/EBPα-positive cells co-stained with Oil red-O were observed only when cells were cultured in adipogenic differentiation medium (Fig. 6A). When cells cultured in adipogenic differentiation medium were double-stained with anti-C/EBPα and anti-ED1 antibodies, C/EBPα-positive cells with lipid droplets were neither stained with anti-ED1 (Fig. 6B) nor anti-ED2 antibodies (data not shown), thus indicating that these C/EBPα/lipid droplet-positive cells are indeed mature adipocytes and not macrophages.
The presence of insulin is required for adipogenic stimulation-induced PPARγ2 expression in cells from control TA muscle but is not required in cells from BPVC-treated TA muscle Pref-1 is known to be abundantly expressed in preadipocytes, and its expression becomes undetectable when cells have
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Fig. 5 – Time course of PPARγ expression in the culture of cells from BPVC-treated TA muscle. Cells were cultured with or without adipogenic stimulation. (A) PPARγ-immunostaining of cells cultured in adipogenic differentiation medium. Black arrowheads indicate lipid droplets/PPARγ double-positive cells. (B) Percentages of PPARγ-positive cells. The data are expressed as mean ± SE (n = 3). (C) RT-PCR analysis of PPARγ2 expression.
differentiated into adipogenic lineage; thus, Pref-1 is suggested to play an important role in maintenance of the undifferentiated preadipose state [7]. The rapid adipogenic differentiation of cells from BPVC-treated TA muscle shown in Fig. 4 led us to examine if differences in expression of Pref-1 exist between cells from control and BPVC-treated TA muscles. As shown in Fig. 7A, Pref-1 mRNA was expressed in cells from control TA muscle that were cultured for 4 days in 10% FBS/DMEM, but its expression was not detected in cells from BPVC-treated TA muscle. This was further confirmed at the protein level by immunocytochemically staining cells with anti-Pref-1 antibody. No cells from BPVC-treated TA muscle showed positive staining whereas some cells from untreated TA muscle showed positive cytoplasmic staining of Pref-1 (Fig. 7B). Considering the possibility if BPVCtreatment itself would alter Pref-1 expression, cells from untreated TA muscle were exposed to BPVC (0.075 mM and 0.0075 mM) and cultured for 3 days but no alteration of Pref-1 expression as revealed by immunocytochemistry was noted (data not shown). These results suggest that rapid adipogenic differentiation of cells from BPVC-treated TA in response to adipogenic stimulation might be attributed to the lack of Pref-1 expression. The negative effect of Pref-1 on adipogenic differentiation has been shown to be blocked by insulin [31]. Our results, demonstrating that Pref-1 is expressed in cells from control TA muscle but not in cells from BPVC-treated TA muscle, and cells from BPVC-treated TA muscle differentiate into adipocytes rapidly upon adipogenic stimulation, led us to predict that cells from BPVC-treated TA muscle are not insulin dependent. To test this possibility, cells isolated from control and BPVCtreated TA muscle were cultured in adipogenic differentiation
medium with or without insulin for 4 days; then PPARγ2 gene expression was examined (Fig. 7C). In the presence of insulin, cells from both control and BPVC-treated TA muscle exhibited PPARγ2 expression. However, in the absence of insulin, only cells from BPVC-treated TA muscle expressed PPARγ2, and its expression level was similar to that observed in cells cultured in the presence of insulin. Therefore, this result indicated that PPARγ2 expression was not insulin dependent in the cells from BPVC-treated TA muscle.
Discussion The major finding of the present study is that experimentally induced degenerative/regenerative changes in skeletal muscle lead to increased adipogenicity of the cells in skeletal muscle. In addition, it was suggested that this increased adipogenicity not only involves a mere increase in the number of cells having adipogenic potential, but also contributes to progression of these cells toward adipogenic differentiation as was indicated by the lack of Pref-1 expression in the cells from BPVC-treated TA muscle. Based on several observations of pathological changes in skeletal muscle involving adipose tissue development [1–4,6], we hypothesized that degenerative/regenerative changes in skeletal muscle could lead to increased adipogenicity of skeletal muscle cells. The most prominent example of adipose tissue development in skeletal muscle is often seen in an advanced stage of DMD in which repeated degeneration/ regeneration of muscle fibers are ongoing [3,4]. In addition, adipose tissue development in skeletal muscle is also observed in denervation-induced atrophy [2] and sarcopenia
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Fig. 6 – Time course of C/EBPα expression in the culture of cells from BPVC-treated TA muscle. Cells were cultured with or without adipogenic stimulation. (A) C/EBPα-immunostaining and Oil Red-O staining. Black and white arrowheads indicate C/EBPα/Oil Red-O double-positive cells and C/EBPα-positive but Oil Red-O-negative cells, respectively. (B) Double-immunostaining for C/EBPα and ED1. Note that C/EBPα-positive cells with lipid droplets are negative for ED1 (black arrowheads).
(age-related loss of muscle mass) [4], and in both cases, degenerative changes of muscle fibers were reported [32,33]. Similar observations were also made in several experimental animal models. For example, adipose tissue development in skeletal muscle is observed in mice deficient of desmin, one of the intermediate filaments present in muscle fibers. Loss of desmin may result in the vulnerability of muscle fibers, leading to their rapid degeneration [34], and is also observed in rabbit muscle denervated for a long period [35]. Therefore, from these observations and the results obtained in the present study, we concluded that the degenerative/regenerative change in skeletal muscle leads to increased adipogenicity of skeletal muscle cells. Skeletal muscle contains several types of progenitor cells that can give rise to adipocytes [36]. These progenitor cells include muscle satellite cells that reside beneath the basal lamina of muscle fibers [20] and CD34+/CD45− cells (SK34 cells) that reside in the interstitial spaces of skeletal muscle [21]. Both types of cells are capable of differentiating into adipocytes [21–23,37,38]. Interestingly, spontaneous adipogenic differentiation of satellite cells emanating from single muscle fibers has been reported [22,37,38]. However, in the present study, no cells from either control or BPVC-treated TA muscles, both likely to contain satellite cells, differentiated into adipocytes unless they were cultured in adipogenic differentiation medium. Considering the difference between the two experimental settings, i.e., culturing satellite cells attached on fibers, and culturing cells isolated from fibers, attachment to muscle fibers for an appropriate period before
emanating might be required for spontaneous adipogenic differentiation of satellite cells. The exact origin of the in vitro differentiated adipocytes derived from control and BPVC-treated TA muscles observed in the present study is unknown at this time. In the present study, we used Pref-1 expression to predict the preadipogenic state of the cells since Pref-1 is known to be expressed in preadipocytes [7], and there are very few markers to predict the preadipogenic state of the cells prospectively. However, Pref-1 expression is not necessarily restricted to preadipocytes. In fact, Pref-1 expression has been shown in a variety of fetal tissues [39]. In addition to its negative effect on adipogenic differentiation, Pref-1 has been reported to inhibit osteogenic differentiation of human mesenchymal cells [40]. Pref-1 expression in adult skeletal muscle after a single bout of exercise has been localized to cells in satellite cell position [41]. Thus, from these studies, Pref-1 is now suggested to have a role in restricting cells to an undifferentiated state [40]. Therefore, we are not able to conclude whether the cells from control TA muscle expressing Pref-1 were preadipocytes and/or another undifferentiated cells. Primary mouse embryonic fibroblasts and several established preadipocyte cell lines can be induced to differentiate into adipocytes when cultured in adipogenic differentiation medium, which typically includes insulin (or insulin growth factor-I (IGF-I)) [9,17]. In the present study, a lack of Pref-1 expression in cells from BPVC-treated TA muscle was seen. We suggest that rapid adipogenic differentiation of cells from
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Fig. 7 – RT-PCR analyses of Pref-1 and PPARγ2, and immunocytochemistry of Pref-1. (A) Mononucleated cells from BPVC-treated or untreated (control) TA muscles were cultured for 4 days in 10% FBS/DMEM, and Pref-1 gene expression was examined. (B) Mononucleated cells from BPVC-treated or untreated (control) TA muscles were cultured for 3 days in 10% FBS/DMEM, and immunocytochemically stained with anti-Pref-1. (C) Mononucleated cells from BPVC-treated or untreated (control) TA muscles were cultured in adipogenic differentiation medium with or without insulin for 4 days, and PPARγ2 gene expression was examined. In both A and C, HPRT was used as an internal control.
BPVC-treated TA in response to adipogenic stimulation may be attributed to this lack of Pref-1 expression. As described above, because of difficulty in directly correlating Pref-1 expression and identification of cells, we had alternatively looked at the difference in insulin dependency of the cells. Our aim was to examine the adipogenicity of the cells regardless of their origin since insulin is known to bypass Pref-1 mediated inhibition of adipogenic differentiation [31]. As expected, cells from control TA muscle required the presence of insulin in adipogenic differentiation medium for their PPARγ2 expression, while cells from BPVC-treated TA muscle did not. Additionally, the lack of effect of BPVC on Pref-1 expression in the cells from control TA muscle in vitro excluded the possibility that BPVC itself had caused any alteration on their adipogenic differentiation potential. Therefore, these results may suggest that Pref-1 expression is downregulated when cells having adipogenic potential are exposed to a degenerative/regenerative environment in skeletal muscle. So far, the factor(s) that induces an increased adipogenicity of the cells in response to BPVC treatment is unknown in the present study. Wnt family proteins are known to inhibit adipogenesis [42], and secreted frizzled-related proteins (sFRPs) that bind to Wnt proteins to inhibit their activity have been shown to induce adipogenesis when applied to preadipocyte culture [43]. The increased adipogenic potential of myoblasts as a function of age has been shown by Taylor-Jones et al. [44], and they indicated the decreased Wnt-10b mRNA expression in myoblasts from aged mice. The same group [45] also revealed that myoblasts from Wnt-10b deficient mice show increased adipogenicity. Akimoto et al. [46] reported that decreased adipogenic potential with increased expression of Wnt-10b mRNA was
noted in mechanically stretched C2C12 cells and the stretchinduced inhibition of adipogenesis was abolished by the presence of sFRP2. In addition, sFRP1 and sFRP2 mRNA expressions have been shown to peak between days 2 and 3 of the cardiotoxin-induced muscle regeneration [47]. Therefore, one of the intriguing possibilities is that alteration of Wnt signaling pathway in skeletal muscle in response to BPVC treatment might have lead to increased adipogenicity of the cells. This issue is currently under investigation in our laboratory. In conclusion, we have shown that degenerative/regenerative changes in skeletal muscle fibers result in increased adipogenicity of cells in skeletal muscle, and suggested that this increased adipogenicity may be attributed to adipose tissue development in skeletal muscle as is seen in some skeletal muscle pathology. Muscle derived progenitor cells are anticipated to be a cellular source for clinical applications in skeletal muscle pathology [37]. In this respect, as a next step, it is of great importance to clarify the tissue environments that would lead them to differentiate into unfavorable cell types such as adipocytes; thereby, precise control of their myogenic differentiation would be achieved. Therefore, further studies to clarify the exact mechanisms of adipose tissue development as well as identifying the factor(s) involved in these processes are required.
Acknowledgments This work was supported by grants to M. Nishihara from the Japan Society for the Promotion of Science, and to K. Yamanouchi from Ministry of Health, Labor and Welfare of
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Japan and Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).
[20] [21]
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
Increased adipogenicity of cells from regenerating skeletal muscle Keitaro Yamanouchi ⁎, Erica Yada, Naomi Ishiguro, Tohru Hosoyama, Masugi Nishihara Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
ARTICLE INFORMATION
ABS T R AC T
Article Chronology:
Adipose tissue development is observed in some muscle pathologies, however,
Received 24 December 2005
mechanisms that induce accumulation of this tissue as well as its cellular origin are
Revised version received
unknown. The adipogenicity of cells from bupivacaine hydrochloride (BPVC)-treated and
4 April 2006
untreated muscle was compared in vitro. Culturing cells from both BPVC-treated and
Accepted 6 April 2006
untreated muscles in adipogenic differentiation medium (ADM) for 10 days resulted in the
Available online 5 June 2006
appearance of mature adipocytes, but their number was 3.5-fold higher in cells from BPVCtreated muscle. Temporal expressions of PPARγ and the presence of lipid droplets during
Keywords:
adipogenic differentiation were examined. On day 2 of culture in ADM, only cells from BPVC-
Skeletal muscle
treated muscle were positive both for PPARγ and lipid droplets. Pref-1 was expressed in cells
Adipocytes
from untreated muscle, whereas its expression was absent in cells from BPVC-treated
Adipose tissue development
muscle. In ADM, the presence of insulin, which negates an inhibitory effect of Pref-1 on
Adipogenesis
adipogenic differentiation, was required for PPARγ2 expression in cells from untreated
Progenitor cells
muscle, but not for cells from BPVC-treated muscle. These results indicate that BPVCinduced degenerative/regenerative changes in muscle lead to increased adipogenicity of cells, and suggest that this increased adipogenicity not only involves an increase in the number of cells having adipogenic potential, but also contributes to the progression of these cells toward adipogenic differentiation. © 2006 Elsevier Inc. All rights reserved.
Introduction In some pathological conditions, increased adipose tissue development (accumulation of adipocytes) is observed in skeletal muscle [1,2]. Adipose tissue development in skeletal muscle is widely recognized as one of the hallmarks of pathological changes seen in Duchenne muscular dystrophy (DMD) [3,4]. It has been described that in advanced cases of DMD, repeated degeneration and regeneration of skeletal muscle fibers finally result in loss of muscle fibers, and this is correlated with an accumulation
of adipocytes in the area where muscle fibers are lost [5]. In addition, the accumulation of adipocytes in skeletal muscle often coincides with sarcopenia (loss of skeletal muscle mass due to atrophy of muscle fibers) [6]. Although mechanisms that induce the accumulation of adipocytes, as well as the cellular origins of adipocytes in skeletal muscle, are unclear, the fact that adipose tissue development is associated with degenerative/regenerative or atrophic changes in skeletal muscle fibers suggests that these changes in skeletal muscle fibers could lead to increased adipogenicity of the cells in skeletal muscle.
⁎ Corresponding author. Fax: +81 3 5841 8017. E-mail address: [email protected] (K. Yamanouchi). 0014-4827/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2006.04.014
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Adipogenic differentiation is under the control of several genes such as preadipocyte factor-1 (Pref-1) [7], peroxisome proliferator activating receptor-γ (PPARγ) [8–11] and CCAAT/ enhancer binding protein-α (C/EBPα) [12]. Pref-1 is a member of the epidermal growth factor (EGF)-like protein family and is abundantly expressed in preadipocytes, whereas its expression in differentiating adipocytes is undetectable [7]. Forced expression of Pref-1 was reported to inhibit adipogenesis; accordingly, Pref-1 was suggested to play an important role in the maintenance of the undifferentiated preadipose state [7]. Terminal differentiation of preadipocytes to adipocytes is believed to be under the control of PPARγ and C/EBPα [13–15]. It has been accepted that once activated, PPARγ and C/EBPα cross-regulate each other to maintain their gene expressions [16,17], and both alone or in cooperation with each other can induce terminal differentiation of adipocytes [9,10,18,19]. Skeletal muscle contains several types of progenitor cells including satellite cells that reside beneath the basal lamina of muscle fiber [20] and CD34+/CD45− cells (SK34 cells) that reside in interstitial spaces of skeletal muscle [21]. Among these cells, recent observation indicates that satellite cells possess the capability to be myogenic stem cells, as well as multipotent stem cells, since satellite cells being cultured in the presence of specific cell lineage inducing cocktails can differentiate into several types of mesenchymal lineages, such as osteocytes and adipocytes [22,23]. In addition, Sk34 cells can be divided into two populations, namely side population (SP) cells and main population (MP) cells, based on the efflux of fluorescent dye Hoechst 33342. Both SP-Sk34 and MP-Sk34 cells also possess adipogenic potential [21]. Bupivacaine hydrochloride (BPVC), a local anesthetic, is widely used for studies on skeletal muscle regeneration. It is known to induce widespread degeneration and necrosis, followed by regeneration of skeletal muscle fibers, without affecting satellite cell viability [24]. A number of studies have been carried out on the BPVC-induced model of skeletal muscle degeneration/regeneration [25–27]. We hypothesized that degenerative/regenerative changes in skeletal muscle could lead to increased adipogenicity of cells in skeletal muscle. Therefore, we have employed a BPVCinduced model of skeletal muscle degeneration/regeneration, and compared the adipogenicity of cells derived from BPVCtreated muscle to those from untreated muscle by culturing them in adipogenic differentiation medium.
Materials and methods Animals and skeletal muscle regeneration Adult male rats (6 to 9 months) of the Wistar Imamichi strain were used throughout this study. They were bred in our laboratory and housed in a room at 23°C with 14 h of light and 10 h of darkness (lights on at 0500 h). Food and water were provided ad libitum. Under light ether anesthesia, 100 μl of bupivacaine hydrochloride (BPVC; 0.75% in saline) was injected into the tibialis anterior (TA) muscles of both legs. Two days after BPVC injection, they were used for isolation of mononucleated cells as described below. As a control, mononucleated cells were
obtained from a rat without BPVC treatment, since BPVC injection into the TA muscle of one side might affect the cells in the TA muscle of the other side. All animal experiments performed in this study were according to the Guideline for the Care and Use of Laboratory Animals, The University of Tokyo.
Isolation and culture of mononucleated cells from skeletal muscle Procedures, previously described by Allen et al. [28] for isolating skeletal muscle satellite cells, were applied to isolate mononucleated cells from skeletal muscle. Briefly, rats injected with BPVC (BPVC-treated) or not injected (control) were sacrificed by inhalation of carbon dioxide gas. The TA muscles were excised, trimmed of fat and connective tissue, hand-minced with scissors and digested for 1 h at 37°C with 1.25 mg/ml pronase (protease; Sigma, MO). Cells were separated from muscle fiber fragments and tissue debris by differential centrifugation and plated on poly-L-lysine and fibronectin-coated dishes in Dulbeccos' modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 50 U/ml penicillin, 50 μg/ml streptomycin and 50 μg/ml gentamicin (10%FBS/DMEM). Typically, a 24-well culture plate was used, and cells were plated at a density based on the starting tissue weight when the cells were isolated (0.5 g of muscle tissues per one well/500 μl 10% FBS/DMEM). Since BPVC treatment is known to increase the cell yield per gram tissue weight [29], thus the initial number of cells plated differed between BPVCtreated and control rats. Cultures were maintained in a humidified atmosphere of 5% CO2 at 37°C. For experiments in which cells were cultured in adipogenic differentiation medium, freshly isolated cells were initially plated in 10% FBS/DMEM and cultured for one day to ensure cell attachment onto the culture surface. Medium was replaced with adipogenic differentiation medium one day after plating (designated as day 0). Adipogenic differentiation medium consisted of the following: from day 0 to day 2, 10% FBS/DMEM supplemented with insulin (1 μg/ml), dexamethazone (0.1 μg/ ml), isobutylmethylxanthine (IBMX; 27.8 μg/ml ) and troglitazone (10 μM; kindly donated by Sankyo Co. Ltd.), from day 2 to day 4, 10% FBS/DMEM supplemented with insulin and troglitazone, and thereafter, 10% FBS/DMEM supplemented only with troglitazone. Medium was replaced every other day until the days indicated in the results. Cells not induced to adipogenically differentiate were cultured in 10% FBS/DMEM only. Additionally, experiment to examine if the difference in the initial cell density between BPVC-treated and control rats could affect the overall results in the present study was performed. In this experiment, cells isolated from BPVC-treated and control TA muscles were plated at same density (6.7 × 105 cells/cm2). One day after plating, cells were fed with adipogenic differentiation medium and cultured for 2 days. The cells were then immunocytochemically stained with anti-PPARγ (described below).
Immunocytochemistry Single staining of MyoD, PPARγ and C/EBPα Unless otherwise stated, all procedures were performed at room temperature (RT). Cultured cells were fixed with 4% paraformaldehyde (PFA) in phosphate buffered-saline (PBS) for 15 min
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(for MyoD and PPARγ) or with ice-cold methanol at − 20°C (for C/ EBPα) for 10 min. Cells were washed with PBS and incubated for 15 min in 5% normal goat serum (NGS) and 0.6% hydrogen peroxide in PBS (blocking solution) to block non-specific binding of antibodies and to quench endogenous peroxidase activity. For MyoD and PPARγ staining, 0.1% triton X-100 was also included in the blocking solution. After washing with PBS, mouse anti-MyoD (1:100 diluted with 5% NGS in PBS; clone 5.8A, Novocastra, Newcastle upon Tyne, UK), anti-PPARγ (1:100 diluted with 5% NGS in PBS; E-8, sc-7273, Santa Cruz, CA) or rabbit anti-C/EBPα antibody (1:200 diluted with 5% NGS in PBS; 14AA, sc-61, Santa Cruz, CA) was applied, and the cells were incubated for 3 h. After washing cells with PBS again, horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Simple stain rat MAX-PO for rabbit IgG; Nichirei, Tokyo, Japan) or HRP-conjugated anti-mouse IgG (Simple stain rat MAX-PO for mouse IgG; Nichirei) was added and incubated for 1 h. Cells were then washed with PBS, and signals were visualized by incubating cells in diaminobenzidine (0.5 mg/ml) and hydrogen peroxide (0.03%) in PBS. In some experiments, cell nuclei were counterstained with hematoxylin and/or cells were doublestained with Oil Red-O (described below) after immunostaining. When quantitative analyses on immunopositive cells and lipid droplet-positive cells were performed, total number of cells and number of positive cells obtained from 10 different fields randomly chosen microscopically using a 40× objective were counted. Cell numbers and the percentages of positive cells were then averaged for the triplicate culture wells. Typically, 150 to 800 cells were counted per well.
Single staining of Pref-1 All procedures were performed at RT. Cultured cells were fixed with 4% PFA in PBS for 15 min, washed with PBS, and incubated for 15 min in 5% NGS in PBS (blocking solution) to block non-specific binding of the antibody. After washing with PBS, rabbit anti-Pref-1 (anti-DLK) antibody (1:100 diluted with 5% NGS in PBS; H-118, sc-25437, Santa Cruz) was applied, and cells were incubated for 3 h. After washing with PBS again, cells were incubated for 1 h in AlexaFluor 594 conjugated-antirabbit IgG (1:200 diluted with 5% NGS in PBS; Invitrogen, CA). Cells were washed with PBS, and nuclei were counterstained with Hoechst 33258. Observations were made using a fluorescence microscope equipped with a digital camera (DP70, Olympus, Tokyo, Japan).
Double staining of C/EBPα and ED1 Unless otherwise stated, all procedures were performed at RT. Cultured cells were fixed with ice-cold methanol at −20°C for 10 min, washed with PBS, and incubated for 15 min in 5% NGS in PBS (blocking solution) to block non-specific binding of the antibodies. After washing with PBS, a mixture of rabbit anti-C/ EBPα antibody (1:200 diluted with 5% NGS in PBS) and mouse anti-ED1 (1:400 diluted with 5% NGS in PBS; BMA Biomedicals AG, Rheinstrasse, Switzerland) was applied, and cells were incubated for 3 h. After washing with PBS again, cells were incubated for 1 h in a mixture of AlexaFluor 488 conjugatedanti-rabbit IgG (1:400 diluted with 5% NGS in PBS; Invitrogen) and AlexaFluor 594 conjugated-anti-mouse IgG (1:400 diluted with 5% NGS in PBS; Invitrogen). Cells were washed with PBS, and nuclei were counterstained with Hoechst 33258. Observa-
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tions were made using a fluorescence microscope equipped with a digital camera.
Oil Red-O staining Cultured cells were fixed with 4% PFA in PBS for 10 min, washed with PBS and stained in Oil Red-O solution (2:3 mixture of 0.5% (w/v) Oil Red-O in 2-isopropanol and distilled water) for 8 min, then washed with PBS. In some experiments, quantitative analyses of Oil Red-O stained cells were performed. Ten days after culture, when cells were at 100% confluency, six different fields randomly chosen under the microscope using a 20× objective were photographed, and areas occupied with Oil RedO-positive cells were quantified using NIH Image software (ver. 1.62, NIH). Values representing incidence of adipogenesis (where applicable) were expressed as the mean pixel measurements for the triplicate culture wells.
RT-PCR Total RNA was isolated from cultured cells at the indicated time points using Trizol reagent. The cDNA was synthesized from 2 μg of total RNA by SuperScript II (Invitrogen) using oligo-dT primer. The PCR was performed on mRNA transcripts of ap2, Pref-1, PPARγ2 and hypoxanthine guanine phosphoribosyltransferase (HPRT), using αTaq polymerase (Bionex, Seoul, Korea). Primer sets for ap2, Pref-1, PPARγ2 and HPRT, annealing temperatures, as well as cycle numbers for corresponding primer sets are as follows: For ap2: Forward 5′-AGCTTGTCTCCAGTGAAAAC-3′, Reverse 5′-GAAGTCACGCCTTTCATAAC-3′, annealing temperature 55°C, 32 cycles. For Pref-1: Forward 5′-GGAAGGCTGGGACGGGAAAT-3′, Reverse 5′- GACACTCGAAGCTCACCTGG-3′, annealing temperature 58°C, 32 cycles. For PPARγ2: Forward 5′-TTCGCTGATGCACTGCCTAT-3′, Reverse 5′-GCCAACAGCTTCTCCTTCTC-3′, annealing temperature 58°C, 30 cycles. For HPRT: Forward 5′-GCTGGTGAAAAGGACCTCT-3′, Reverse 5′-CACAGGACTAGAACRYCTGC-3′, annealing temperature 58°C, 29 cycles (R stands for A or G; Y stands for C or T; these were included so that this primer set could be used both for rat and mouse samples in our laboratory). Annealing temperatures and cycle numbers were determined so that the amplification reactions were within the linear range. The PCR products were visualized with ethidium bromide after agarose-gel electrophoresis. Quantitative analysis on the mRNA expression levels was performed on the photo of the gel using NIH image software, and the data are indicated as pixel.
Statistical analyses Each experiment was repeated two to three times, and only representative data are shown. The data are expressed as means ± SE. Statistical analyses were performed using commercial software (StatView, ver. 4.5, Abacus Concepts, Inc., Berkley, CA). Student's t test or one-way ANOVA followed by Bonferroni's test were used to evaluate statistical
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differences among the groups. P values less than 0.05 were considered as statistically significant.
Results BPVC-treatment increases the number of cells capable of differentiating into mature adipocytes In order to determine if BPVC-treatment increases the total number of cells capable of differentiating into mature adipocytes, mononucleated cells were isolated from control and BPVC-treated TA muscles and cultured with or without adipogenic stimulation for 10 days. After 10 days of culture, when cells under any condition were nearly 100% confluent, Oil Red-O staining was used to determine the degree to which cells had differentiated into mature adipocytes . In the absence of adipogenic stimulation, no cells were stained with Oil Red-O. On the other hand, in the presence of adipogenic stimulation, some cells from control TA muscle acquired a mature adipocyte-like phenotype, as represented by Oil Red-O staining, while the incidence of adipogenesis was significantly increased (>3.5-fold; P < 0.05) in cells from BPVC-treated TA muscle (Fig. 1). In addition, mRNA expression levels of ap2, a marker representing for the presence of mature adipocytes, were compared between the cells from BPVC-treated and control TA muscles cultured with adipogenic differentiation medium for 4 days. As shown in Fig. 2, more lipid droplet-positive cells were seen in the cells from BPVC-treated TA muscle and accordingly, mRNA expression level of ap2 was also increased (>2-fold; P < 0.01) in the cells from BPVC-treated TA muscle compared to those from control TA muscle. These results indicate that in response to BPVC-treatment, the number of cells capable of differentiating into mature adipocytes increases.
Expressions of MyoD, PPARγ and C/EBPα in cells from control and BPVC-treated TA muscles Mononucleated cells were prepared from BPVC-treated or control TA muscle and their expressions of MyoD, PPARγ and
C/EBPα were examined on day 2 after plating in 10% FBS/ DMEM. Nearly all cells (>95%) from control TA muscle exhibited positive MyoD staining, whereas only a portion of cells from BPVC-treated TA muscle were positive for MyoD (Fig. 3A), indicating the presence of non-myogenic cells. This was in agreement with the previous result shown by Molnar et al. [29]. PPARγ and C/EBPα were negligible in cells from control TA muscle (Fig. 3B and C), indicating the absence of differentiated adipocytes in control TA muscle. Cells from BPVC-treated TA muscle were also negative for PPARγ (Fig. 3B), but rich in C/EBPα-positive cells (Fig. 3C). However, a vast majority of C/EBPα-positive cells were either co-stained with anti-ED1, a marker for invading macrophages [30] (Fig. 3C) or anti-ED2, a marker for resident macrophages [30] (data not shown). Thus, these results indicate that without adipogenic stimulation, there are no differentiated adipocytes in cells from BPVCtreated TA muscle.
Rapid differentiation into adipocytes of cells from BPVC-treated TA muscle upon adipogenic stimulation Although there were no differentiated adipocytes in cells from BPVC-treated TA muscle, the results shown in Fig. 1 and 2 indicated increased adipogenicity of cells from BPVC-treated TA muscle upon adipogenic stimulation. In order to know whether this is due to the mere increase in number of cells that are capable of differentiating into adipogenic lineage in response to BPVC treatment, and/or is attributed to the appearance of cells that are proceeding to adipogenic differentiation, cells from control and BPVC-treated TA muscle were cultured in adipogenic differentiation medium for 2 days, and PPARγ, C/EBPα expressions and the presence of lipid droplets were examined. As expected and shown in Fig. 4A, number of cells from BPVC-treated TA muscle on day 2 of culture was 2.4fold higher (p<0.01) than that from control TA muscle. No cells positive for lipid droplets/PPARγ were seen in the culture from control TA muscle (Fig. 4B). On the other hand, 7.9% of the cells from BPVC-treated TA muscle were double-positive for PPARγ and lipid droplets (Fig. 4B). Similar result was obtained when the initial cell density was matched between the cells form
Fig. 1 – Appearance of Oil Red-O positive cells in response to adipogenic stimulation in vitro. Mononucleated cells from BPVC-treated or untreated (control) TA muscles were cultured with or without adipogenic stimulation for 10 days. Graphed data indicates the quantitative analysis on the number of Oil Red-O positive cells. Data are expressed as mean ± SE (n = 3).
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Fig. 2 – RT-PCR analysis on ap2 mRNA expression levels in the cells from BPVC-treated and control TA muscles. Cells were cultured with adipogenic stimulation for 4 days. (A) Lipid droplet-positive cells were indicated by white arrowheads. (B) Quantitative analysis of ap2 mRNA expression levels. The data are expressed as means ± SE (n = 3). There was no statistical difference in HPRT mRNA expression levels. neg, negative control (PCR without template).
Fig. 3 – Immunocytochemical analyses of myogenic and adipogenic markers in mononucleated cells from BPVC-treated or untreated (control) TA muscles. Cells were cultured in 10% FBS/DMEM for 2 days. (A) MyoD. Black and white arrowheads indicate positive and negative cells, respectively. (B) PPARγ. No positive cells were seen. (C) C/EBPα. Cells were also double-stained for C/EBPα and ED1. Note that most C/EBPα-stained cells are positive for ED1. Black arrowheads in panel C (middle) indicate C/EBPα-positive cells. Black and white arrowheads (C, right) indicate C/EBPα-positive but ED1-negative cells and C/EBPα/ED1 double-positive cells, respectively.
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Fig. 4 – Quantitative analyses of adipogenic differentiation of cells from BPVC-treated and untreated (control) TA muscle on day 2 of culture with adipogenic stimulation. Cells were cultured in adipogenic differentiation medium for 2 days. (A) Total number of cells. (B) Percentages of lipid droplet/ PPARγ double-positive cells. (C) C/EBPα-immunostaining and Oil Red-O staining. Black and white arrowheads indicate C/EBPα/Oil Red-O double-positive cell and C/EBPα-positive but Oil Red-O-negative cell, respectively. Graphed data (A and B) are expressed as mean ± SE (n = 3).
BPVC-treated and control TA muscles (PPARγ-positive cells, 1.8 ± 0.9% (control, n = 3) vs. 21.7 ± 0.9% (BPVC-treated, n = 3)). C/ EBPα-positive cells were present in both cultures (Fig. 4C), but the percentage of C/EBPα-positive cells was much higher in cells from BPVC-treated TA muscle (26.9%) than in cells from control TA muscle (1.5%). This might be due to the presence of macrophages present in the culture from BPVC-treated TA muscle as was shown in Fig. 3C. However, among the cells from BPVC-treated TA muscle that are positive for C/EBPα, some cells co-stained with Oil Red-O, suggesting the presence of mature adipocytes. Taken together, these results suggest that increased adipogenicity of cells from BPVC-treated TA muscle involves progression of these cells toward adipogenic differentiation, though without adipogenic stimulation, they are neither expressing PPARγ nor C/EBPα as yet.
Time course of PPARγ and C/EBPα expression in cells from BPVC-treated TA muscle with or without adipogenic stimulation To examine thoroughly expressions of PPARγ and C/EBPα during adipogenic differentiation of cells from BPVC-treated TA muscle, time course studies were carried out. For PPARγ expression, no cells were positive on day 0 of culture (Fig. 5A and B). PPARγpositive cells appeared on day 2 of culture when the cells were cultured in adipogenic differentiation medium, confirming the result shown in Fig. 4B, and the percentage of PPARγ-positive
cells was maintained on day 4 of culture (Fig. 5A and B). On the other hand, no PPARγ-positive cells were observed up to 4 days when they were cultured in 10% FBS/DMEM (Fig. 5B). Since PPARγ2 is known to be a highly specific marker of adipocytes [11], its mRNA expression was determined by means of RT-PCR. In agreement with the result obtained by PPARγ-immunocytochemistry, the expression level of PPARγ2 mRNA was negligible on day 0 of culture, but it became apparent on days 2 and 4 of culture in response to adipogenic stimulation (Fig. 5C). C/EBPα-positive cells were observed at all time points examined regardless of adipogenic stimulation (Fig. 6A). However, C/EBPα-positive cells co-stained with Oil red-O were observed only when cells were cultured in adipogenic differentiation medium (Fig. 6A). When cells cultured in adipogenic differentiation medium were double-stained with anti-C/EBPα and anti-ED1 antibodies, C/EBPα-positive cells with lipid droplets were neither stained with anti-ED1 (Fig. 6B) nor anti-ED2 antibodies (data not shown), thus indicating that these C/EBPα/lipid droplet-positive cells are indeed mature adipocytes and not macrophages.
The presence of insulin is required for adipogenic stimulation-induced PPARγ2 expression in cells from control TA muscle but is not required in cells from BPVC-treated TA muscle Pref-1 is known to be abundantly expressed in preadipocytes, and its expression becomes undetectable when cells have
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Fig. 5 – Time course of PPARγ expression in the culture of cells from BPVC-treated TA muscle. Cells were cultured with or without adipogenic stimulation. (A) PPARγ-immunostaining of cells cultured in adipogenic differentiation medium. Black arrowheads indicate lipid droplets/PPARγ double-positive cells. (B) Percentages of PPARγ-positive cells. The data are expressed as mean ± SE (n = 3). (C) RT-PCR analysis of PPARγ2 expression.
differentiated into adipogenic lineage; thus, Pref-1 is suggested to play an important role in maintenance of the undifferentiated preadipose state [7]. The rapid adipogenic differentiation of cells from BPVC-treated TA muscle shown in Fig. 4 led us to examine if differences in expression of Pref-1 exist between cells from control and BPVC-treated TA muscles. As shown in Fig. 7A, Pref-1 mRNA was expressed in cells from control TA muscle that were cultured for 4 days in 10% FBS/DMEM, but its expression was not detected in cells from BPVC-treated TA muscle. This was further confirmed at the protein level by immunocytochemically staining cells with anti-Pref-1 antibody. No cells from BPVC-treated TA muscle showed positive staining whereas some cells from untreated TA muscle showed positive cytoplasmic staining of Pref-1 (Fig. 7B). Considering the possibility if BPVCtreatment itself would alter Pref-1 expression, cells from untreated TA muscle were exposed to BPVC (0.075 mM and 0.0075 mM) and cultured for 3 days but no alteration of Pref-1 expression as revealed by immunocytochemistry was noted (data not shown). These results suggest that rapid adipogenic differentiation of cells from BPVC-treated TA in response to adipogenic stimulation might be attributed to the lack of Pref-1 expression. The negative effect of Pref-1 on adipogenic differentiation has been shown to be blocked by insulin [31]. Our results, demonstrating that Pref-1 is expressed in cells from control TA muscle but not in cells from BPVC-treated TA muscle, and cells from BPVC-treated TA muscle differentiate into adipocytes rapidly upon adipogenic stimulation, led us to predict that cells from BPVC-treated TA muscle are not insulin dependent. To test this possibility, cells isolated from control and BPVCtreated TA muscle were cultured in adipogenic differentiation
medium with or without insulin for 4 days; then PPARγ2 gene expression was examined (Fig. 7C). In the presence of insulin, cells from both control and BPVC-treated TA muscle exhibited PPARγ2 expression. However, in the absence of insulin, only cells from BPVC-treated TA muscle expressed PPARγ2, and its expression level was similar to that observed in cells cultured in the presence of insulin. Therefore, this result indicated that PPARγ2 expression was not insulin dependent in the cells from BPVC-treated TA muscle.
Discussion The major finding of the present study is that experimentally induced degenerative/regenerative changes in skeletal muscle lead to increased adipogenicity of the cells in skeletal muscle. In addition, it was suggested that this increased adipogenicity not only involves a mere increase in the number of cells having adipogenic potential, but also contributes to progression of these cells toward adipogenic differentiation as was indicated by the lack of Pref-1 expression in the cells from BPVC-treated TA muscle. Based on several observations of pathological changes in skeletal muscle involving adipose tissue development [1–4,6], we hypothesized that degenerative/regenerative changes in skeletal muscle could lead to increased adipogenicity of skeletal muscle cells. The most prominent example of adipose tissue development in skeletal muscle is often seen in an advanced stage of DMD in which repeated degeneration/ regeneration of muscle fibers are ongoing [3,4]. In addition, adipose tissue development in skeletal muscle is also observed in denervation-induced atrophy [2] and sarcopenia
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Fig. 6 – Time course of C/EBPα expression in the culture of cells from BPVC-treated TA muscle. Cells were cultured with or without adipogenic stimulation. (A) C/EBPα-immunostaining and Oil Red-O staining. Black and white arrowheads indicate C/EBPα/Oil Red-O double-positive cells and C/EBPα-positive but Oil Red-O-negative cells, respectively. (B) Double-immunostaining for C/EBPα and ED1. Note that C/EBPα-positive cells with lipid droplets are negative for ED1 (black arrowheads).
(age-related loss of muscle mass) [4], and in both cases, degenerative changes of muscle fibers were reported [32,33]. Similar observations were also made in several experimental animal models. For example, adipose tissue development in skeletal muscle is observed in mice deficient of desmin, one of the intermediate filaments present in muscle fibers. Loss of desmin may result in the vulnerability of muscle fibers, leading to their rapid degeneration [34], and is also observed in rabbit muscle denervated for a long period [35]. Therefore, from these observations and the results obtained in the present study, we concluded that the degenerative/regenerative change in skeletal muscle leads to increased adipogenicity of skeletal muscle cells. Skeletal muscle contains several types of progenitor cells that can give rise to adipocytes [36]. These progenitor cells include muscle satellite cells that reside beneath the basal lamina of muscle fibers [20] and CD34+/CD45− cells (SK34 cells) that reside in the interstitial spaces of skeletal muscle [21]. Both types of cells are capable of differentiating into adipocytes [21–23,37,38]. Interestingly, spontaneous adipogenic differentiation of satellite cells emanating from single muscle fibers has been reported [22,37,38]. However, in the present study, no cells from either control or BPVC-treated TA muscles, both likely to contain satellite cells, differentiated into adipocytes unless they were cultured in adipogenic differentiation medium. Considering the difference between the two experimental settings, i.e., culturing satellite cells attached on fibers, and culturing cells isolated from fibers, attachment to muscle fibers for an appropriate period before
emanating might be required for spontaneous adipogenic differentiation of satellite cells. The exact origin of the in vitro differentiated adipocytes derived from control and BPVC-treated TA muscles observed in the present study is unknown at this time. In the present study, we used Pref-1 expression to predict the preadipogenic state of the cells since Pref-1 is known to be expressed in preadipocytes [7], and there are very few markers to predict the preadipogenic state of the cells prospectively. However, Pref-1 expression is not necessarily restricted to preadipocytes. In fact, Pref-1 expression has been shown in a variety of fetal tissues [39]. In addition to its negative effect on adipogenic differentiation, Pref-1 has been reported to inhibit osteogenic differentiation of human mesenchymal cells [40]. Pref-1 expression in adult skeletal muscle after a single bout of exercise has been localized to cells in satellite cell position [41]. Thus, from these studies, Pref-1 is now suggested to have a role in restricting cells to an undifferentiated state [40]. Therefore, we are not able to conclude whether the cells from control TA muscle expressing Pref-1 were preadipocytes and/or another undifferentiated cells. Primary mouse embryonic fibroblasts and several established preadipocyte cell lines can be induced to differentiate into adipocytes when cultured in adipogenic differentiation medium, which typically includes insulin (or insulin growth factor-I (IGF-I)) [9,17]. In the present study, a lack of Pref-1 expression in cells from BPVC-treated TA muscle was seen. We suggest that rapid adipogenic differentiation of cells from
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Fig. 7 – RT-PCR analyses of Pref-1 and PPARγ2, and immunocytochemistry of Pref-1. (A) Mononucleated cells from BPVC-treated or untreated (control) TA muscles were cultured for 4 days in 10% FBS/DMEM, and Pref-1 gene expression was examined. (B) Mononucleated cells from BPVC-treated or untreated (control) TA muscles were cultured for 3 days in 10% FBS/DMEM, and immunocytochemically stained with anti-Pref-1. (C) Mononucleated cells from BPVC-treated or untreated (control) TA muscles were cultured in adipogenic differentiation medium with or without insulin for 4 days, and PPARγ2 gene expression was examined. In both A and C, HPRT was used as an internal control.
BPVC-treated TA in response to adipogenic stimulation may be attributed to this lack of Pref-1 expression. As described above, because of difficulty in directly correlating Pref-1 expression and identification of cells, we had alternatively looked at the difference in insulin dependency of the cells. Our aim was to examine the adipogenicity of the cells regardless of their origin since insulin is known to bypass Pref-1 mediated inhibition of adipogenic differentiation [31]. As expected, cells from control TA muscle required the presence of insulin in adipogenic differentiation medium for their PPARγ2 expression, while cells from BPVC-treated TA muscle did not. Additionally, the lack of effect of BPVC on Pref-1 expression in the cells from control TA muscle in vitro excluded the possibility that BPVC itself had caused any alteration on their adipogenic differentiation potential. Therefore, these results may suggest that Pref-1 expression is downregulated when cells having adipogenic potential are exposed to a degenerative/regenerative environment in skeletal muscle. So far, the factor(s) that induces an increased adipogenicity of the cells in response to BPVC treatment is unknown in the present study. Wnt family proteins are known to inhibit adipogenesis [42], and secreted frizzled-related proteins (sFRPs) that bind to Wnt proteins to inhibit their activity have been shown to induce adipogenesis when applied to preadipocyte culture [43]. The increased adipogenic potential of myoblasts as a function of age has been shown by Taylor-Jones et al. [44], and they indicated the decreased Wnt-10b mRNA expression in myoblasts from aged mice. The same group [45] also revealed that myoblasts from Wnt-10b deficient mice show increased adipogenicity. Akimoto et al. [46] reported that decreased adipogenic potential with increased expression of Wnt-10b mRNA was
noted in mechanically stretched C2C12 cells and the stretchinduced inhibition of adipogenesis was abolished by the presence of sFRP2. In addition, sFRP1 and sFRP2 mRNA expressions have been shown to peak between days 2 and 3 of the cardiotoxin-induced muscle regeneration [47]. Therefore, one of the intriguing possibilities is that alteration of Wnt signaling pathway in skeletal muscle in response to BPVC treatment might have lead to increased adipogenicity of the cells. This issue is currently under investigation in our laboratory. In conclusion, we have shown that degenerative/regenerative changes in skeletal muscle fibers result in increased adipogenicity of cells in skeletal muscle, and suggested that this increased adipogenicity may be attributed to adipose tissue development in skeletal muscle as is seen in some skeletal muscle pathology. Muscle derived progenitor cells are anticipated to be a cellular source for clinical applications in skeletal muscle pathology [37]. In this respect, as a next step, it is of great importance to clarify the tissue environments that would lead them to differentiate into unfavorable cell types such as adipocytes; thereby, precise control of their myogenic differentiation would be achieved. Therefore, further studies to clarify the exact mechanisms of adipose tissue development as well as identifying the factor(s) involved in these processes are required.
Acknowledgments This work was supported by grants to M. Nishihara from the Japan Society for the Promotion of Science, and to K. Yamanouchi from Ministry of Health, Labor and Welfare of
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Japan and Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).
[20] [21]
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