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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
USDA-ARS, Richard B. Russell Agricultural Research Center, Animal Physiology Research Unit, Athens, GA 30605-2720
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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(C/EBP) and peroxisome proliferator-activated receptor gamma (PPAR
) positive cells in adipose tissue S-V cultures, whereas PPAR-reactive but not C/EBP-reactive cells were increased in muscle S-V cultures treated with 10 µM troglitazone. Additionally, troglitazone treatment did not increase lipid content in s.c. adipose tissue or muscle S-V cell cultures. Cells plated on laminin-precoated culture dishes were used to determine whether troglitazone influenced adipogenesis or myogenesis in cocultures from muscle S-V cells. There was no effect on the number of myotubes or the average number of nuclei per myotube, suggesting myogenesis was not impaired by troglitazone treatment. These results suggest that regulation of intramuscular adipogenesis differs from that of subcutaneous adipogenesis.
Key Words: adipose tissue • cell culture • cell differentiation • fat cell • skeletal muscle • transcription factor
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Van Barneveld, 2003; Wood et al., 1999; Schoonmaker et al., 2004; Poulos and Hausman, 2005). In addition, studies have associated intramuscular adiposity with age, sex, diet, and muscle type (Poulos and Hausman, 2005). However, very little is known about the growth and development of intramuscular adipocytes or the regulation of intramuscular adipogenesis. This lack of information limits the development of methods to regulate growth of these cells and improve marbling fat content of meat animals.
In vitro studies directly comparing dexamethasone (Dex) -induced preadipocyte recruitment in stromal-vascular (S-V) cell cultures from subcutaneous adipose tissue and skeletal muscle indicate that response to Dex may differ between these 2 populations of preadipocytes (Hausman and Poulos, 2004). These studies suggest that metabolic differences may exist between intramuscular adipocytes and adipocytes of other depots. Thus, the aim of this study was to compare thiazolidinedione (TZD) -induced preadipocyte recruitment and differentiation in S-V cell cultures from subcutaneous adipose tissue and muscle. Thiazolidinediones are a class of pharmaceutical agents currently used in the treatment of type 2 diabetes mellitus that are also known as adipogenic compounds (Furnsinn and Waldhausl, 2002). The number of CCAAT/enhancer-binding protein (C/EBP) and peroxisome proliferator-activated receptor (PPAR) reactive cells were quantified because of the known roles of these transcription factors in adipocyte differentiation and the known ligand binding of TZD to PPAR (Sakamoto et al., 2000; Hammarstedt and Smith, 2003). Additionally, AD-3 reactive cells were quantified to examine recruitment of preadipocytes in S-V cell cultures from muscle and adipose tissue. Secondly, this study compared the adipogenic and myogenic response with TZD in cocultures of intramuscular adipocytes and myocytes.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The protocols used in these experiments were approved by the USDA, Richard Russell Center, Agricultural Research Service, Animal Care and Use Committee. Five- to seven-day-old postnatal pigs (C42 x 280; PIC, Franklin, KY) from The University of Georgia swine herd were euthanized using 3 g of sodium thiopental injected intraperitoneally, followed by exanguination. Ventral s.c. adipose tissue and both semitendinosus muscles (STM) were aseptically removed. Primary S-V cells from s.c. adipose tissue and both muscles were isolated using a collagenase digestion procedure as previously described (Hausman and Poulos, 2004). Adipose tissue (SQ) S-V cells were plated at a density of 1 x 105 cells per 35-mm culture dish. To maintain similar times required for cells to reach a confluent density, semitendinosus muscle S-V cells were plated at a density of 2.5 x 105 cells per 35-mm culture dish (Hausman and Poulos, 2005). Adipose tissue and muscle S-V cells were plated on 35-mm culture dishes with proliferation media, which consisted of Dulbecco’s Modified Eagle’s Medium with F12 supplement (DMEM; Sigma Aldrich, St. Louis, MO) and 10 mL of fetal bovine serum/L (FBS; Sigma Aldrich). Dishes were rinsed with DMEM, and 2 mL of treatment media were added to each dish approximately 1 h after plating. Upon reaching confluency, 3 to 4 d after plating, cultures were rinsed with DMEM and given 2 mL of DMEM supplemented with 5 IU of bovine insulin/mL, 5 µg of human transferrin/mL, and 5 ng of selenium/mL (ITS; Sigma-Aldrich), which is a differentiating medium. Throughout the experiment, cultures were maintained in a 37°C environment with 5% CO2.
Dose Response to TZD
Experiment 1 was conducted to determine the effective dose of the TZDs, ciglitazone and troglitazone. In Exp. 1, treatment media included DMEM + 10 mL of FBS/L, supplemented with 0.01% (vol/vol) dimethyl sulfoxide (DMSO) vehicle; 10, 25, or 50 µM troglitazone in DMSO; or 10, 25, or 50 µM ciglitazone in DMSO (BioMol Inc., Plymouth Meeting, PA). Immunocytochemical evaluation for AD-3, a preadipocyte marker, was performed after 3 d of ITS treatment. The AD-3 monoclonal antibody recognizes a cell surface antigen on preadipocytes, before lipid accretion and the development of other markers of overt adipocyte differentiation (Hausman and Richardson, 1998).
Effect of Troglitazone on Adipogenesis in Muscle and Adipose S-V Cell Cultures
Experiment 2 was performed to determine the influence of 10 µM troglitazone on adipogenesis in adipose or muscle S-V cells (n = 4 to 6 pigs). Treatment media included DMEM + 10 mL of FBS/L, supplemented with either 0.01% (vol/vol) DMSO vehicle or 10 µM troglitazone in DMSO. After 3 d in ITS-containing media, cultures were evaluated for AD-3, C/EBP, or PPAR using immunocytochemistry. To quantify lipid content, cultures were also stained with Oil-Red-O (Sigma Aldrich).
Effect of Troglitazone on Myogenesis in Muscle S-V Cell Cultures
Experiment 3 was performed to determine the influence of 10 µM troglitazone on adipogenesis and myogenesis in S-V cells from muscle (n = 5 pigs). As previously described (Hausman and Poulos, 2005), plating cells on 35-mm laminin-precoated culture dishes (BD Biosciences, Bedford, MA) allowed for the coculture of adipocytes and myotubes from muscle. Treatment media included DMEM + 10 mL of FBS/L, supplemented with either 0.01% (vol/vol) DMSO vehicle or 10 µM troglitazone. After 3 d of differentiation in ITS containing media, cultures were evaluated for AD-3, C/EBP, or PPAR using immunocytochemistry. Culture dishes were also stained with Oil-Red-O and hematoxylin to quantify lipid content and myotube formation, respectively.
Immunohistochemistry
As previously described, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min (Hausman, 2003). Cells were either permeabilized with 3% Nonidet P-40 for 20 min for C/EBP and PPAR localization, or were not permeabilized for AD-3 localization. Endogenous peroxides were quenched using a 3% H2O2 solution for 5 min. Cells were then incubated in a humidified chamber for 30 min with primary antibody against C/EBP (1/50, sc61, Santa Cruz Biotechnology Inc., Santa Cruz, CA), PPAR (1/200, sc7273, Santa Cruz Biotechnology), or the AD-3 antibody (1/200). This was followed by incubation with biotinylated anti-mouse or anti-rabbit immunoglobulin G (1/100) for 30 min and extravidin peroxidase for 30 min. Reactivity was visualized using AEC, a colorimetric substrate. Lipid content was quantified using a 60% Oil-Red-O solution for 10 min followed by hematoxylin staining for 2 min, as previously described (Hausman, 1981). Quantification of stained cells (AD-3), nuclei (C/EBP, PPAR), lipid content, or total cell number per field was completed using 4x-magnified fields (Olympus, Melville, NY) and computer-assisted image analysis (Image-Pro Plus, Media Cybernetics, Inc., Silver Spring, MD). Myotube number was manually determined using 3 microscopic fields per culture dish at 10x. A minimum of 3 culture dishes per treatment and stain were evaluated for each experiment.
Statistical Analysis
Data were analyzed by the Proc GLM procedure of SAS (SAS Inst. Inc., Cary, NC). One batch of SQ and 1 batch of STM S-V cells were isolated from each pig and were used for 1 replicate. Each replicate included a minimum of 3 culture dishes for each treatment. In Exp. 1, cell number was tested using a model with main effects of cell source, dose, TZD type, and the interactions of cell source x dose and cell source x TZD type. In Exp. 2, the main effects of cell source, treatment, and their interaction on immunoreactive cell number and fold changes in immunoreactive cell number were tested. Treatment was the main effect tested on adipocyte number and myotube number in Exp. 3. Data are reported as least square means ± SEM and expressed relative to control treatments. Differences were considered significant at P < 0.05 and tendencies at P < 0.10.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Though both skeletal muscle and adipose tissue contain various cell types, it was expected that adipose tissue S-V cell cultures would contain more adipogenic cells than S-V cultures from STM. As expected, preadipocyte number after 3 d in ITS was greater in adipose S-V cultures as compared with muscle S-V cultures (reactive cells per 10x microscopic field; 24.1 ± 16.4 SQ, 9.8 ± 5.5, STM, P < 0.0001) regardless of treatment. Increased preadipocyte number was observed in adipose S-V cultures treated with 10 µM ciglitazone or troglitazone relative to DMSO control treated cultures (reactive cells per 10x microscopic field; 15.3 ± 7.8, DMSO control; 30.5 ± 7.8, ciglitazone; 38.9 ± 7.8, troglitazone; P < 0.05). Additionally, increasing doses in either treatment did not result in further increases in preadipocyte number (Table 1). This is in contrast to muscle S-V cultures, which did not show an increase in preadipocyte number at 10, 25, or 50 µM ciglitazone (Table 1). Regardless of dose, AD-3 reactive cells were approximately 3-fold greater in troglitazone-treated cultures than in DMSO-treated cultures in muscle S-V cultures. Thus, it appears troglitazone appears to be more adipogenic than ciglitazone in both SQ and STM S-V cultures (Table 1). There were no differences in AD-3 reactive cell number in FBS and 0.01% DMSO-treated cells of adipose or muscle S-V cultures (data not shown).
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Experiment 1 showed that the 10 µM troglitazone dose was the lowest TZD dose tested that increased adipogenesis in both adipose and muscle S-V cell cultures. This dose was used in Exp. 2 and 3. In Exp. 2 (Table 2), troglitazone treatment increased fold changes in AD-3 reactive cells in both adipose (1.0 ± 0.3, DMSO; 4.1 ± 0.3, troglitazone; P < 0.0001) and muscle S-V cell cultures (1.0 ± 0.2, DMSO; 3.0 ± 0.2, troglitazone; P < 0.0001). Although both adipose and muscle cell cultures responded to TZD, fold increases in AD-3 reactive cells in adipose cultures were significantly greater than in muscle cultures (P < 0.05). Fold increases in C/EBP were significant in adipose cultures (1.0 ± 0.3, DMSO; 2.5 ± 0.3, troglitazone; P < 0.01) and tended to be significant in muscle cultures (1.0 ± 0.3, DMSO; 1.7 ± 0.3, troglitazone; P = 0.06). Troglitazone treatment increased PPAR reactive cells to approximately 3 times the number of PPAR stained cells in DMSO treated cells in a 20x microscopic field (Table 2). Lipid area per 20x microscopic field and average lipid droplet diameter were not affected by treatment and did not differ between muscle and adipose S-V cell cultures (P > 0.05).
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As expected, laminin induced myotube formation in muscle S-V cell cultures (Figure 1, Hausman and Poulos, 2005). However, there was no significant difference between DMSO and troglitazone treated muscle S-V cell cultures regarding the number of myotubes (myotubes per 10x microscopic field, 4.7 ± 1.9, DMSO; 5.4 ± 1.7, troglitazone). Myotubes were not significantly different in size as determined by the average number of nuclei per myotube (Table 3). The adipogenic response, as quantified by the number of AD-3, C/EBP, and PPAR immunoreactive cells per microscopic field, was not affected by laminin in muscle S-V cell cultures (Table 3). For example, regardless of treatment there were 18.7 ± 6.4 AD-3 reactive cells in a 20x microscopic field of muscle S-V cells on laminin coated culture dishes and 19.4 ± 5.6 AD-3 reactive cells in a 20x microscopic field of muscle S-V cells on uncoated culture dishes (means ± SEM of 3 experiments). However, fold changes in C/EBP reactive cell number tended to be increased by troglitazone in muscle S-V cells plated on uncoated dishes (1.0 ± 0.3, DMSO; 1.8 ± 0.2, troglitazone) but not on laminin-coated dishes (1.0 ± 0.4, DMSO; 1.3 ± 0.5, troglitazone). This trend was more evident with PPAR reactive cell number in muscle S-V cell cultures. Fold changes in PPAR reactive cell number were significant when cells were plated on un-coated culture dishes (1.0 ± 0.8, DMSO; 3.3 ± 0.8; P < 0.05) but not when cells were plated on laminin-coated dishes (1.0 ± 1.4; DMSO; 1.6 ± 1.4, troglitazone). However, the main effect of the interaction between substrata and troglitazone treatment was not significant (Table 3). Lipid content, as assessed by lipid area per 20x microscopic field and average lipid droplet area, were not different between treatments or substrata.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Furnsinn and Waldhausl, 2002). Our primary cultures of S-V cells from muscle are one of the few published models used to investigate intramuscular adipogenesis. Cultures of muscle and adipose tissue from the same animals allow for the in vitro comparison of adipogenesis between these 2 depots. Additionally, coculture of myotubes and adipocytes in muscle S-V cell cultures allows us to investigate the interactions between these 2 cell types that are in spatial proximity in vivo. Previous studies from our laboratory have shown that primary S-V cultures from muscle contain preadipocytes which develop into mature, lipid-containing adipocytes that express PPAR (Hausman and Poulos, 2004). Ciglitazone has also been shown to induce PPAR and glucose transporter 4 (GLUT4) expression, increase insulin stimulated glucose transport and long-chain fatty acid transport in fibroblasts adjacent to skeletal muscle in mice, suggesting that intramuscular preadipocyte proliferation is induced (Lohrke et al., 2000). Thus, it is likely that the increased PPAR expression in muscle cell cultures is actually from adipocytes induced to differentiate with TZD.
Results from Exp. 1 show that the adipogenic response to TZD is greater in adipose S-V cell cultures than in muscle S-V cell cultures as determined by quantification of AD-3 reactive cells. There was approximately a 5-fold increase in adipogenesis with troglitazone in adipose S-V cell cultures, whereas troglitazone increased adipogenesis 3-fold in muscle S-V cell cultures. Differences in relative abundance of PPAR expressing cells between muscle and adipose S-V cell cultures are likely responsible for the greater adipogenic response by adipose S-V cells compared with muscle S-V cells. In particular, PPAR2 expression would be much greater in adipose tissue than muscle because this transcription factor is virtually only expressed by adipocytes. Though expression of PPAR, PPARß/
, and PPAR is low, significant levels of these proteins are present in skeletal muscle (Loviscach et al., 2000). Levels of PPAR, specifically PPAR1, in skeletal muscle are much lower than in adipose tissue. Additionally, troglitazone enhances adipogenesis to a greater extent than ciglitazone in both adipose and muscle S-V cultures. This may be due to the differences in binding affinity and activation for PPAR transcription factors and specific binding among the different compounds within the TZD class of pharmaceutical agents (Berger et al., 1996; Perfetti et al., 1998; Sakamoto et al., 2000).
The 10 µM troglitazone treatment was chosen for the remaining studies because it was the lowest effective dose tested for increasing relative AD-3 number in both muscle and adipose S-V cell cultures. Experiment 2 confirmed that 10 µM troglitazone induces adipogenesis in S-V cultures of both adipose and muscle origin. This experiment showed a 3-fold increase in AD-3 reactive cells in troglitazone treated muscle S-V cultures compared with controls, whereas there was approximately a 4-fold increase in relative AD-3 reactive cells in adipose S-V cultures. Troglitazone treatment increased PPAR reactive cells in adipose tissue and in muscle S-V cultures. Additionally, C/EBP reactive cell number was determined as this transcription factor is involved in adipogenesis and insulin sensitivity but is not thought to be a ligand for TZD. An increase in C/EBP reactive cells due to troglitazone was observed in both adipose S-V cultures and muscle S-V cultures. The lack of difference in lipid content, but increase in the number of AD-3 reactive cells, in Exp. 2 and 3 suggests that troglitazone induces the recruitment of preadipocytes but does not increase lipid accretion. These results agree with results in adipose S-V cell cultures induced to differentiate using troglitazone (Tchoukalova et al., 2000). In summary, it appears that troglitazone increases the number of preadipocytes and adipocytes in both muscle and adipose S-V cultures. However, there is no significant difference in lipid content in these cells after 3 d of insulin. Nonetheless, it appears that adipocytes in both adipose and muscle S-V cultures treated with troglitazone are truly adipocytes with endocrine function. This is supported by the trend toward increased C/EBP reactive cells.
Studies of cultured rat L6 muscle cells showed that TZD increased C/EBP, and IRS-1 gene and protein expression but did not influence adipocyte lipid binding protein, an adipocyte marker (Hammarstedt and Smith, 2003). However, in vitro studies of human skeletal muscle cells from biopsy samples showed that troglitazone treatment increased adipocyte lipid binding protein, PPAR2, and glycerophosphate dehydrogenase gene expression and suppressed myogenic differentiation (Kausch et al., 2001). Induction of adipocyte marker proteins suggested the possibility of transdifferentiation of myocytes to adipocytes (Hu et al., 1995; Grimaldi et al., 1997). However, the authors did not determine if their biopsy samples were free of adipocytes (Kausch et al., 2001). Thus, the differentiation of adipocytes in these muscle samples, and the resulting changes in gene expression, cannot be excluded. The current study clearly shows troglitazone-enhanced expression of adipocyte markers in the absence of an influence on myogenesis. In particular, preadipocyte recruitment, as assessed by an increase in AD-3 reactive cell number, was markedly enhanced in the absence of any change in myotube size or number. Thus, it is possible that recruitment or differentiation of adipogenic precursors per se, or both, in biopsy samples was induced by troglitazone, resulting in the shift toward a more adipogenic culture (Kausch et al., 2001). Our in vitro system is advantageous over other coculture systems because the cells coexist in vivo and are cultured with minimal presence of external products. In a recent review, it was suggested that communication exists between skeletal muscle and adipose tissue to regulate energy homeostasis (Argiles et al., 2005). Although various cell types are present in both of these tissues, cross talk between muscle fibers and intramuscular adipocytes could be substantial.
Muscle extracts inhibit adipocyte differentiation in bovine intramuscular S-V cells (Sato et al., 1996), suggesting that the interaction between skeletal muscle and adipose tissue may influence the development of these tissues. We examined this possibility in Exp. 3 since adipogenesis was induced in muscle S-V cell cultures in the presence (laminin-coated dishes) and absence (uncoated dishes) of myotube development. The presence of myotubes had no influence on TZD induced increases in AD-3, C/EBP, or PPAR immunoreactive cell numbers. This suggests that in vitro myotube formation and intramuscular adipogenesis occur independently. However, it should be noted that this system lacks some of the communication that may occur in vivo. These include neural and electrical stimulation of muscle or the release of myokines that may result in communication with intramuscular adipocytes to provide energy substrates to contracting skeletal muscle. It is possible that differences are not observed under basal conditions, represented by this in vitro system. Furthermore, it is also possible that the low number of myotubes in laminin-coated dishes may have obscured any influence of myotube-derived factors on adipogenesis.
These studies show that TZD have physiologically relevant effects on both muscle and adipose tissue S-V cells and thus have the potential to modify intramuscular or marbling adipogenesis. Differences in response to ciglitazone and troglitazone in muscle but not adipose S-V cultures suggests that intramuscular and adipose tissue preadipocytes differ in regards to either PPAR content or PPAR affinity for TZD. Troglitazone induced marbling adipogenesis but did not influence myogenesis. Therefore, troglitazone treatment in vivo may be useful to enhance marbling in the growing animal. Finally, primary muscle S-V cell cultures can be used to screen other TZD or comparable agents for their capability to influence marbling adipogenesis.
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Footnotes |
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2 Correspondence: ghausman{at}saa.ars.usda.gov
Received for publication August 4, 2005. Accepted for publication December 7, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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