J. Anim Sci. 2008. 86:E226-E235. doi:10.2527/jas.2007-0450
© 2008 American Society of Animal Science
Application of cellular mechanisms to growth and development of food producing animals1,2
K. Y. Chung and
B. J. Johnson3
Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506
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Abstract
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Postnatal skeletal muscle growth is a result of hypertrophy of existing skeletal muscle fibers in food producing animals. Accumulation of additional nuclei, as a source of DNA, to the multinucleated skeletal muscle fiber aids in fiber hypertrophy during periods of rapid skeletal muscle growth. Muscle satellite cells are recognized as the source of nuclei to support muscle hypertrophy. Exogenous growth-enhancing compounds have been used to modulate growth rate and efficiency in meat animals for over a half century. In cattle, these compounds enhance efficiency of growth by preferentially stimulating skeletal muscle growth compared with adipose tissue. There are 2 main classes of compounds approved for use in cattle in the United States, anabolic steroids and β-adrenergic agonists (β-AA). Administration of both trenbolone acetate and estradiol-17β, as implants, increased carcass protein accumulation 8 to 10% in yearling steers. Muscle satellite cells isolated from steers implanted with trenbolone acetate/ estradiol-17β had a shorter lag phase in culture compared with satellite cells isolated from control steers. Collectively, these data indicate that activation, increased proliferation, and subsequent fusion of satellite cells in muscles of implanted cattle may be an important mechanism by which anabolic steroids enhance muscle hypertrophy. Oral administration of β-AA to ruminants does not alter DNA accumulation in skeletal muscle over a typical feeding period (28 to 42 d). Enhanced muscle hypertrophy observed due to β-AA feeding occurs by direct, receptor-mediated changes in protein synthesis and degradation rates of skeletal muscle tissue. Proper timing of anabolic steroid administration when coupled with β-AA feeding could result in a synergistic response in skeletal muscle growth due to the effects of anabolic steroids at increasing satellite cell activity, which then can support the rapid hypertrophic changes of the muscle fiber when exposed to β-AA. At the same time each of these classes of compounds are stimulating lean tissue deposition, they appear to repress adipogenesis in meat animals. Increased knowledge of the mechanism by which growth promotants regulate lean tissue deposition and adipogenesis in meat animals will allow for effective application of these techniques to optimize lean tissue growth and minimize the negative effects on meat quality.
Key Words: adipose tissue • β-adrenergic agonist • anabolic steroid • skeletal muscle • transdifferentiation
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INTRODUCTION
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Growth promotants, such as steroidal implants and β-adrenergic agonists, shift nutrient utilization toward carcass lean tissue deposition at the expense of adipose tissue. Previous work with implants containing trenbolone acetate (TBA) and estradiol-17β (E2) increased carcass weight 18 to 27 kg but had no effect on fat thickness as compared with nonimplanted fed the same number of days (Johnson et al., 1996a
). We observed an initial burst of protein gain during the first 40 d after implantation with TBA and E2 that resulted in carcasses from implanted steers with approximately 10 to 12% more carcass protein than carcasses from nonimplanted steers fed the same number of days. This study indicated that differences in carcass protein mass at the end of the feeding period after implantation with TBA and E2 may be largely due to significant increases in muscle deposition during the first 40 d after implantation. This has served as a biological basis of why some delayed implant or low-dose implant programs in yearling cattle have been successful at attenuating the decrease in quality grade at harvest. By delaying this rapid increase in lean tissue accretion until later in the feeding period, intramuscular adipogenesis may initiate at a greater rate. However, instead of focusing on the shift of nutrient utilization during periods of growth promotion or lack thereof, the focus of this discussion will be at the cellular level, particularly focusing on muscle-derived progenitor cells. It is our hypothesis that growth promotion affects the direction in which certain nondifferentiated, stem-cell-like mesodermal cells proceed. An increased understanding of cellular mechanisms affected by growth promotion then in turn can help explain shifts in nutrient utilization by the whole animal. The objectives of this review are to offer brief overviews of skeletal muscle and adipose tissue growth and development, the process of transdifferentiation between 2 cell types, and conclude with how growth promotants may direct a nondifferentiated cell to become a certain lineage.
SKELETAL MUSCLE GROWTH
The individual muscle fiber is considered the cellular unit of skeletal muscle tissue. The postnatal skeletal muscle fiber has several distinguishing characteristics compared with cells that make up other tissues. The muscle fibers, as well as nuclei within each fiber, are postmitotic, having lost the ability to divide. Additionally, muscle fiber number is fixed at birth in most meat animals. In order to sustain postnatal muscle hypertrophy, the muscle fiber needs an external source of DNA. The DNA accumulation responsible for postnatal muscle hypertrophy is highly correlated with muscle growth rate (Trenkle et al., 1978). In fact, 60 to 90% of DNA located within mature skeletal muscle fibers is accumulated during postnatal growth (Allen et al., 1979). Muscle satellite cells are now known to be the source of DNA responsible for postnatal muscle hypertrophy (Figure 1). By supplying more DNA to the individual fiber, there is more "machinery" available to ultimately synthesize a greater amount of protein within each fiber; thus, the positive relationship between DNA content in the fiber and rate of muscle growth in cattle.
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Figure 1. The role of muscle satellite cells in supporting postnatal muscle growth. Satellite cells lie in close proximity to the existing fiber and under appropriate stimuli can undergo cell proliferation. Eventually, the majority of these cells will fuse into the existing fiber, thus donating their DNA to support skeletal muscle hypertrophy. The process of transdifferentiation would convert muscle satellite cells to cells capable of accumulating lipid droplets in the cytosol.
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Satellite cells are mononucleated cells located between the basal lamina and sarcolemma of the muscle fiber (Mauro, 1961). Moss and Leblond (1970) determined there were 2 types of nuclei within the basement membrane of the muscle fiber that were distinguishable from one another. Following the labeling of nuclei by a single [3H]-thymidine injection, male rats were killed at different time intervals and the tibialis anterior muscle was removed for autoradiographic analysis. The results of this study showed that the true muscle nuclei were not labeled at 1 h following injection, indicating they were not actively dividing. However, the nuclei within the basement membrane were labeled, indicating the satellite cells were able to synthesize DNA and divide. Moss and Leblond (1970) also reported that the number of labeled true muscle nuclei was increasing, whereas the number of labeled satellite cells decreased. This led to the conclusion that the source of labeled nuclei being counted within the fiber over the 72-h time frame were, in fact, that of satellite cells that were dividing and incorporating into the existing muscle fibers. Once the satellite cells fused with the existing fiber and donated their nuclei, they, in turn, lost their proliferative capacity (Moss and Leblond, 1971). These studies confirmed the postmitotic nature of true muscle nuclei and the importance of the muscle satellite cell in postnatal skeletal muscle growth.
The necessity of satellite cells in postnatal muscle growth is well understood, although there are still limitations to the degree of DNA accretion at later stages of muscle growth. In a newborn animal, 30% of muscle nuclei are satellite cells, but the number decreases between 2 and 10% in mature animals, thus showing that the actual number of satellite cells decreases with age (Cardasis and Cooper, 1975). This becomes a challenge in optimizing skeletal muscle hypertrophy in more mature cattle due to the small population of progenitor cells available to contribute to the existing muscle fibers. Not only is there a reduction in satellite cell number, but those cells still present also withdraw from the proliferative state of the cell cycle and enter 0 (a state of quiescence), which leads to a growth plateau (Cardasis and Cooper, 1975). In order to maintain the satellite cell population necessary to support muscle hypertrophy in mature animals, the cells in quiescence must be activated to allow them to progress through the cell cycle and contribute nuclei to the existing muscle fiber. Once quiescent satellite cells have been activated, there is a need for growth factors capable of stimulating satellite cell proliferation and subsequent differentiation. Insulin-like growth factor-I and fibroblast growth factor-2 are known as progression factors due to their ability to aid in progressing cells through the cell cycle. Both growth factors are potent stimulators of satellite cell proliferation (Allen and Rankin, 1990; Johnson and Allen, 1990). However, IGF-I is unique in skeletal muscle in that it also promotes muscle cell differentiation, whereas fibroblast growth factor-2 inhibits differentiation (Allen and Boxhorn, 1989; Allen and Rankin, 1990). Members of the transforming growth factor-β superfamily are also capable of regulating satellite cell activity. These growth factors are considered negative regulators of skeletal muscle in that they inhibit proliferation and differentiation (Allen and Rankin, 1990). One member of the transforming growth factor-β superfamily responsible for negative regulation of skeletal muscle is myostatin, also known as growth and differentiation factor 8 (McPherron et al., 1997). Myostatin is responsible for double muscling observed in cattle due to a mutation in the myostatin gene (McPherron and Lee, 1997). This embryonic mutation leads to a greater number of muscle fibers in each muscle, as witnessed in double-muscled cattle. This important discovery has led to evaluation of the possible use of myostatin in therapeutic settings, such as for treatment of muscular dystrophy, as well as future use in growth promoting systems for meat animals. The regulation of growth factor-mediated changes in satellite cell proliferation and differentiation is controlled by a family of transcription factors called myogenic regulatory factors (MRF). The MRF family includes Myo D, Myf-5, myogenin, and MRF-4. In concert, these transcription factors determine the fate of mononucleated cells, which finally become muscle cells. However, recent novel findings in a rodent model report that resident, specialized muscle cells can be converted in vivo and in vitro into the adipocytes that make up the intramuscular fat (marbling) within the muscle (Wada et al., 2002; Singh et al., 2003).
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REGULATION OF ADIPOGENESIS IN BEEF CATTLE
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Marbling is often defined as the adipose tissue within muscle bundles or intramuscular adipose tissue. It is generally recognized that marbling is the last adipose tissue to be deposited on a finishing beef animal, although adipose tissue starts to accumulate in the early weaning periods (Harper and Pethick, 2004). Marbling score continues to be the single most important factor for determining carcass quality in the United States and abroad. However, there has been a marked decrease in USDA carcass quality grades of beef carcasses during the last 3 decades, although the average USDA yield grade has not changed during that period.
Cattle can accumulate adipose tissue nearly indefinitely, and strong evidence exists to indicate that some portion of the increase in adiposity in the mature animal is derived from proliferation and differentiation of a preexisting pluripotent fibroblasts. There is evidence to indicate that some portion of fat infiltration in skeletal muscle may arise from interconversion of muscle satellite cells into adipocytes under conditions of disuse or enervation (Wada et al., 2002). Beef cattle provide an especially suitable model for investigations of this process because they are noted for vast amounts of marbling adipose tissue accumulation within their muscles (Lunt et al., 1993). Many researchers have studied cell size in intramuscular adipose tissues. Intramuscular adipose tissue has larger cells per unit mass, smaller mean cell diameter, and smaller mean cell volume than subcutaneous and perirenal adipose tissue (Smith and Crouse, 1984). These authors demonstrated that different regulatory mechanisms were present in subcutaneous and intramuscular adipose tissue. For example, acetate contributed 70 to 80% of the acetyl units for in vitro lipogenesis in subcutaneous adipose tissue, but only 10 to 25% in intramuscular adipose tissue. Likewise, glucose contributed 1 to 10% of the acetyl units in subcutaneous adipose tissue and 50 to 75% in intramuscular adipose tissue. These results showed that stromal-vascular cells within intramuscular adipose tissue were quite proliferative and used glucose for making acetyl units in adipose tissue. Also, these results confirmed that the lipogenic metabolism in intramuscular adipose tissue resembled that of myogenic metabolism in skeletal muscle tissue. Differences among breeds contribute to size and amounts of adipocyte in intramuscular adipose tissue. In Wagyu steers, which are high-marbling beef cattle, marbling adipocytes are smaller and exhibit twice the rate of DNA synthesis as marbling adipocytes from Angus steers at the same physiological maturity (May et al., 1994, Chung et al., 2007). Wagyu steers contained intramuscular adipose tissues that result not only from hypertrophy of adipocytes, but also by hyperplasia of adipocytes at any time (May et al., 1994). These findings contributed to the understanding of intramuscular adipogenesis and the need to approach it at a fundamental, cellular basis.
Recent research findings support the concept of 2 different lineages making up backfat or marbling. This is based on the finding that these 2 types of adipocytes are derived from 2 distinctly different origins. It appears that adipocytes that are found in subcutaneous adipose tissue are derived from brown adipose tissue that was present at birth in calves (Alexander et al., 1975). However, recent novel findings in a rodent model report that resident, specialized muscle cells can be converted into the adipocytes that make up the intramuscular fat (i.e., marbling) within the muscle in vivo and in vitro (Wada et al., 2002; Singh et al., 2003).
The 3T3-L1 cell line is one of the most well-characterized and reliable models for studying the conversion of preadipocytes into adipocytes. Upregulation of genes important during differentiation is summarized in Figure 2. When injected into mice, 3T3-L1 preadipocytes differentiate and form fat pads that are indistinguishable from normal adipose tissue. In culture, differentiated 3T3-L1 preadipocytes possess most of the structural characteristics of adipocytes from animal tissue. The formation and appearance of developing fat droplets also mimic live adipose tissue (Green and Kehinde, 1974). Confluent 3T3-L1 preadipocytes can be differentiated synchronously by a defined adipogenic component. Maximal differentiation is achieved upon treatment with the combination of insulin, a glucocorticoid, an agent that elevates intracellular cAMP levels and fetal bovine serum (Cornelius et al., 1994). Insulin is known to act through the IGF-I receptor. Insulin-like growth factor I can be substituted for insulin in the adipogenic cocktail. Dexamethasone, a synthetic glucocorticoid agonist, is traditionally used to stimulate the glucocorticoid receptor pathway. Methylisobutylxanthine, a cAMP phosphodiesterase inhibitor, is traditionally used to stimulate the cAMP-dependent protein kinase pathway. These adipogenic components are commonly abbreviated MDI (methylisobutylxanthine, dexamethasone, and insulin). Approximately 24 h after induction by MDI, differentiating preadipocytes undergo a postconfluent mitosis and subsequent growth arrest (Bernlohr et al., 1985). The cells undergo at least 1 round of DNA replication and cell division. By d 2 of differentiation, the cells complete the postconfluent mitosis and enter into an unusual growth arrest called GD (Scott et al., 1982). This terminal mitosis is believed necessary to unwind DNA, allowing transcription factors access to regulatory response elements present in genes involved in modulating the mature adipocyte phenotype (Cornelius et al., 1994). After the growth arrest, cells are committed to becoming adipocytes. The growth arrest is required for subsequent differentiation. Growth-arrested cells begin to express late markers of differentiation at d 3. These late markers consist of lipogenic and lipolytic enzymes, as well as other proteins responsible for modulating the mature adipocyte phenotype. The cells then round up, accumulate fat droplets, and become terminally differentiated adipo-cytes by d 5 to 7.
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Figure 2. The role of transcription factors related to preadipocyte differentiation. Transcription factors (e.g., ADD1, SREBP1, and C/EBP), regulated by specific ligands, are involved in triggering preadipocyte differentiation and expression of adipocyte-specific genes (e.g., FAS, SCD, TNF , Leptin) by binding to transcription control elements (here labeled as "Factors binding site"). Transdifferentiation converts muscle-derived cells to preadipocytes. Early, Intermediate, and Late = stage of adipocyte differentiation. Functional activation is the final stage of adipocyte differentiation.
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Adipose differentiation processes are genetically regulated by diverse hormonal and nutritional factors. The CCAAT/enhancer-binding protein β (C/EBPβ) and the peroxisome proliferators-activated receptor 
(PPAR), transcriptional factors expressed during early and intermediate stage of adipocyte differentiation induce preadipocytes to mature adipocytes. The CCAAT/enhancer-binding protein β protein expressed in early stage of adipose differentiation induces mesen-chymal precursor cells into preadipocytes (Wu et al., 1995). Among the PPAR, PPARis expressed primarily in adipose tissue and is induced preadipocyte differentiation with retinod x receptor as a heterodimer (Figure 2
). There is a report that PPARis expressed in the myoblast cell line and can suppress the muscle specific transcription factors, Myf5, MyoD, myogenin, and MRF4 (Hu et al., 1995). These results suggested that adipogenic transcription factors are also involved in satellite cell differentiation.
Stearoyl-Co A desaturase (SCD) gene expression can be used as a later marker for adipocyte differentiation (Figure 2). Expression of the SCD gene is induced by growth factors and hormones, and SCD promotes de novo fatty acid synthesis not only in the adipocyte, but also in the muscle tissue (Chang et al., 1992; Miyazaki and Ntambi, 2003). Gene expression and enzymatic activity of the SCD in bovine adipose tissues are indicators of fat softness and important aspects of meat quality (Smith et al., 1998; Chung et al., 2007). Stearoyl-Co A desaturase is an endoplasmic reticulum-anchored enzyme that converts palmitoyl-CoA and stearoyl-CoA to palmitoleoyl-CoA and oleoyl-CoA catalyzed by the NADH- and O2-dependent desaturation of saturated fatty acid. (Miyazaki and Ntambi, 2003). A high dietary carbohydrate and insulin induced SCD gene expression through sterol regulatory element binding protein 1 cascade (Shimano, 2001). Monounsaturated fatty acid are used as major precursors for the synthesis of various lipid forms, including triacylglycerol, phospholipids, cholesterol ester, and wax esters. Oleic acid, a major monounsaturated fatty acid in animal adipose tissue synthesized by SCD, is the active precursor for acyl-CoA cholesterol acyltransferase in cholesterol ester biosynthesis and diacylglycerol acyltransferase in triacylglycerol synthesis. Oleic acid also has been reported to regulate cell development and differentiation through control of membrane fluidity and signal transduction (Ntambi, 1999; Miyazaki et al., 2001). activity of SCD affects not only the fatty acid composition in plasma membranes but also lipid metabolism in adipose tissue. Therefore, C/EBPβ, PPAR, and SCD not only activate many lipogenic genes and control adipogenesis but are also involved in the central role of adipose differentiation.
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CONVERSION OF PRIMITIVE CELLS TO A DIFFERENT FATE: TRANSDIFFERENTIATION
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There are several in vitro studies that have demonstrated a role for adipogenic transcriptional factors, PPARand C/EBP (Table 1, Figure 2), at inducing adipocyte number in the skeletal muscle in cattle and pigs (Torii et al., 1998; Poulos and Hausman, 2006). When exposed to thiazolidinedione (TZD), these 2 adipogenic transcriptional factors are expressed during the transdifferentiation process of myoblasts, These factors did not express when myoblasts were treated under optimal myogenic differentiation conditions (Hu et al., 1995). Thiazolidinediones and long chain fatty acids can induce transdifferentiation of myoblasts to adipocytes (Hu et al., 1995; Teboul et al., 1995; Grimaldi et al., 1997, De Coppi et al., 2006). Thiazolidinediones, used as antidiabetic agents, improve insulin sensitivity and glucose uptake by activating GLUT4 (Mukherjee et al., 2000). The physiological activity of TZD can affect the balance of muscle and adipose stromal-vascular cell and, consequently, affect intramuscular adipogenesis (Poulos and Hausman, 2006). Thiazolidinedione-mediated adipogenesis of intramuscular adipose tissue may contribute to enhanced quality of beef cattle. However, there are other physiological activities for using TZD that have been reported. Thiazolidinediones can trans differentiate bone marrow cells to adipocyte and unbalance of the adipogenic activity in bone marrow, which causes anemia (Gimble et al., 1996). Also, TZD may decrease the skeletal muscle mass by suppression of myogenic gene expression (Singh et al., 2003). Torii et al. (1998) reported that fibroblast-like cells, resident in bovine skeletal muscle, could be converted to adipoblasts when exposed to the TZD, T-174. Interestingly, the endogenous ligand of PPAR, prostaglandin J2, could not induce the bovine-derived fibroblast cells to become adipocytes (Torii et al., 1998), as has been reported in rodent models (Forman et al., 1995). This suggests potential species differences.
A more recent report investigated the effect of a potent TZD, ciglitizone, on transdifferentiation of porcine muscle satellite cells to adipocytes (Poulos and Hausman, 2006). Under normal myogenic culture conditions, porcine muscle satellite cells became multinucleated myotubes indicating normal myogenic differentiation. Exposure of these muscle satellite cells to ciglitizone completely ameliorated fusion (formation of multinu-cleated myotubes) and caused formation of cells containing lipid droplets indicative of conversion of satellite cells to adipoblasts. Further investigation revealed that in the ciglitizone-treated groups, expression of C/ EBP and PPARwere upregulated. Consequently, the expression of C/EBP and PPARwas sufficient to block muscle differentiation and result in transdifferentiation of muscle satellite cells to adipocytes. Our preliminary data support that conversion of myogenic satellite cells to adipogenic cells occurs (Figure 3). Long chain fatty acids and TZD definitely improved adipogenic activity during satellite cell differentiation. Oil red-O staining demonstrated multilipid droplets not only in the myotubes, but also in the cytosol of individual muscle-derived cells. Taken together, these results show the profound effect TZD and long chain fatty acids can have on conversion of muscle cells to adipocytes. The next generation of research will need to begin investigating the effect of these products on in vivo changes in lipid content of muscles.
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Figure 3. Photomicrographs of morphological changes related to in vitro fate of bovine muscle-derived cells, which are induced by various factors and stained with oil red-O. Transdifferentiation is the ability of presumed muscle cells to convert to adipocytes.
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GROWTH PROMOTANTS AND CELLULAR RESPONSES
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Insulin-like growth factor I was shown to be a potent stimulator of protein synthesis in skeletal muscle and at the same time can reduce the rate of protein degradation (Florini et al., 1996). Previous research demonstrated that administration of a combined TBA and E2 implant resulted in increased circulating IGF-I and IGF-I mRNA levels in the longissimus muscles of implanted steers compared with nonimplanted steers 30 to 40 d after implantation (Johnson et al., 1996b, 1998b; Dunn et al., 2003; White et al., 2003). In addition, Pampusch et al. (2003) reported that IGF-I mRNA abundance in longissimus muscle biopsy samples from implanted steers were greater than those of nonimplanted steers as quickly as 12 d after implantation. These results indicate that the muscle of implanted steers may produce more IGF-I than that of nonimplanted cattle. Additionally, circulating levels of IGF-I will be greater in sera from cattle implanted with TBA and E2 compared with nonimplanted cattle (Johnson et al., 1996b). Taken together, these effects of implanting will have positive effects on enhancing protein accretion in existing skeletal muscle fibers.
For the increase in protein mass to be sustained long-term in skeletal muscle, eventually the fiber will need more "machinery" or added DNA to aid in the process of protein synthesis. As discussed above, this is an important role of the muscle satellite cell. Satellite cells lie between the basal lamina and sarcolemma of individual muscle fibers. They are capable of proliferating and ultimately fuse into the adjoining fiber to donate their nuclei to support the ramped-up protein synthesis. Consequently, factors that can impact rate of satellite cell incorporation into existing fibers will have a positive impact on postnatal muscle hypertrophy. Administration of TBA and E2 to yearling steers resulted in an increase in the number of actively proliferating satellite cells within 35 d of implantation (Johnson et al., 1998a). This is important in light of the fact that only a small number of satellite cells are present at this time in yearling cattle. In addition many of these satellite cells have become quiescent or left the cell cycle. We feel that an important mode of action of anabolic steroid-mediated muscle hypertrophy involves altering the activity of muscle satellite cells. It is thought that the enhanced IGF-I production by the muscle fiber after administration of the steroid implants mediated the increased proliferative activity of these satellite cells. In addition, in vitro studies have revealed that TBA and E2 can directly increase the rate of cell proliferation of cultured satellite cells isolated from bovine skeletal muscle (Kamanga-Sollo et al., 2004). Based on the previous discussion, increased proliferative activity of satellite cells should enhance the rate of muscle growth in cattle. Taken together, these findings strongly support a mechanism for steroid implant-induced muscle growth in beef cattle that involves increases in the local production of muscle IGF that, in turn, enhances satellite cell activity and, consequently, increases skeletal muscle growth.
If we assume that there are a small number of these progenitor cells present in more mature bovine skeletal muscle, administration of steroidal implants will potentially increase proliferative capacity and ultimately induce differentiation of the daughter cells within the existing muscle fibers. A recent report further showed that as compounds, like anabolic steroids, were causing progenitor cells to go down the myogenic pathway, they were also blocking their entry to the adipogenic pathway. Singh et al. (2003) used the pluripotent immortalized cell line, C3H 10T1/2, to investigate the direct effect of androgens on myogenic and adipogenic differentiation. Interestingly, the number of myogenic cells and myosin protein levels increased in a dose-dependent fashion in response to both testosterone and dihydrotes-tosterone addition. At the same time these 2 steroids decreased the number of adipocytes formed by the C3H 10T1/2 cells and downregulated both C/EBP- and PPAR-protein expression. These profound effects were blocked by a specific androgen receptor antagonist, bicalutamide, indicating the steroids were mediating these cell fates through the androgen receptor on the pluripotent cells. Although conducted with rodent pluripotent cells in a cell culture model, these results increase our understanding of the potential effects of anabolic steroids used in implants on the push of primitive muscle-derived cells to stay muscle cells and not become adipocytes. Thus, this offered us a cellular explanation of how growth promotion could positively impact skeletal muscle growth and simultaneously inhibit marbling.
Another recent report challenges the effects of TBA and E2 administration on inhibiting markers of adipose conversion. Smith et al. (2007) reported that administration of 2 Synovex Plus implants (200 mg of TBA and 28 mg of estradiol benzoate) to both steers and heifers did not alter transcription of mRNA for important markers of adipogenesis, such as acetyl CoA carboxylase, stearyl CoA desaturase, and lipoprotein lipase, at the end of the 140-d feeding period. Although the authors did not analyze changes over a time course following implanting, one could hypothesize that changes at the end of the feeding period may not be reflective of what occurred immediately following implanting. Interestingly, Smith et al. (2007) demonstrated that in steers the number of intramuscular adipocytes per gram of tissue was greater in implanted cattle compared with nonimplanted cattle. In addition, this response only occurred for the intramuscular adipocytes and not subcutaneous adipocytes. In heifers, these differences only tended to be different but paralleled the response observed in steers. One could hypothesize that the administration of the implant earlier in the feeding period engaged the primitive cells to proliferate, albeit most went on to become muscle, and that the pool available to transdifferentiate could have been larger due to implanting. Therefore, at the end of the feeding period when steroid levels waned, these cells became intramuscular adipocytes within the muscle.
It appears that progestins may have opposite effects of androgens and estrogens in skeletal muscle. In a study by Sissom et al. (2006), the addition of melengestrol acetate (MGA) to cultured bovine satellite cells resulted in a dose-dependent decrease in [3H]-thymidine incorporation when supraphysiological and physiological concentrations of MGA were used. Furthermore, in the experiments utilizing C2C12 myoblasts, both MGA and progesterone addition resulted in significant reductions in [3H]-thymidine incorporation when IGFBP-3-stripped media was utilized. In order to examine the mechanism by which MGA and progesterone reduced [3H]-thymidine incorporation rate in C2C12 myoblasts, the progestin antagonist RU 486 was utilized (Sissom et al., 2006). Progesterone activity is normally inhibited by RU 486 through inhibition of progesterone binding to the nuclear progesterone receptor, to which RU 486 is an antagonist (Sager et al., 2003). However, in these experiments, the addition of RU 486 to cultures treated with MGA or progesterone did not block the reduction in [3H]-thymidine incorporation (Sissom et al., 2006). Interestingly, RU 486 added alone to C2C12 myoblasts resulted in a significant reduction in [3H]-thymidine incorporation similar to MGA-and progesterone-treated cultures. Possibly, RU 486 can bind to plasma membrane progesterone receptor and function as a weak agonist.
Nongenomic actions of steroids do not involve binding to the classic nuclear receptor and, therefore, may not be affected by inhibitors of that mechanism, such as RU 486 (Stormshak and Bishop, 2008). Additionally, these responses are very rapid and involve second messenger systems, such as cyclic AMP or intracellular Ca2+. These data support the hypothesis that the reduction in [3H]-thymidine incorporation rate observed in C2C12 myoblasts treated with MGA or progesterone may be mediated through a nongenomic mechanism, which provides further insight into the direct actions of progestins on skeletal muscle. It is interesting that many nutritionists feel that inclusion of MGA may improve marbling scores. The fact it appears to have antianabolic properties in muscle cell cultures may imply that it can stimulate transdifferentiation of muscle cells to adipocytes; however, more research needs to be conducted to determine the role of progestins in transdifferentiation.
We have addressed the effects of anabolic steroid growth promotion on cellular conversion; now, what impact might feeding approved β-adrenergic agonists have on cellular transdifferentiation? One of the most pronounced effects of feeding a β-adrenergic agonist to ruminants is the preferential dramatic increase in skeletal muscle mass, cross-sectional area of individual muscles, or both (Beermann et al., 1987; Miller et al., 1988; Smith et al., 1995). Due to the dramatic increase in skeletal muscle hypertrophy following β-adrenergic agonist administration to ruminants, one would expect satellite cell proliferation and subsequent fusion of the satellite cells to provide a source of DNA to support the rapid changes in muscle mass, similar to action of steroid implants. However, the majority of previous work indicated that during the 3 to 5 wk of β-adrenergic agonist stimulated muscle hypertrophy, no change in number of nuclei occurred (Beermann et al., 1987; Kim et al., 1987; O’Connor et al., 1991). A constant amount of DNA (i.e., nuclei number), coupled with rapid changes in muscle mass and, consequently, protein accumulation, results in lower DNA concentration of individual muscles in β-adrenergic agonistfed animals compared with untreated controls (Beermann et al., 1987). Because DNA accumulation during rapid periods of muscle hypertrophy does not occur due to feeding a β-adrenergic agonist, many researchers have focused on the direct effect of β-adrenergic agonists binding to their receptors (β-adrenergic receptors) affecting rate of protein synthesis, protein degradation, or both (Sissom et al., 2007; Winterholler et al., 2007). Skeletal muscle in cattle has been shown to have abundant numbers of β-adrenergic receptors on the cell surface (Sillence and Matthews, 1994). Previous research has shown that many β-adrenergic agonists are capable of increasing protein synthesis and decreasing protein degradation (Kim et al., 1987). The net effect of these changes are dramatic changes in accretion of protein within skeletal muscle tissue. It appears that β-adrenergic agonists cause existing nuclei within the muscle fiber to become much more efficient at increasing muscle protein accumulation without the support of additional DNA from satellite cells. However, over a course of 3 to 5 wk it becomes difficult for skeletal muscle to sustain this level of fiber hypertrophy without additional DNA, and consequently, responsiveness to the β-adrenergic agonists is dampened. These results indicate that administration of a β-adrenergic agonist to cattle may have minimal effects on primitive cell activity.
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CONCLUSIONS
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Commonly used growth promotants, such as steroidal implants and β-adrenergic agonists, have recently been implicated as one contributing factor that has led to reduced marbling scores in beef cattle. These compounds are effective at improving lean tissue deposition and significantly improving feed efficiency in cattle. An increased understanding of how these agents affect cellular aspects of growth and development of skeletal muscle and adipose tissue will allow cattle feeders, consultants, and researchers to initiate intervention strategies to ameliorate the reduced marbling scores. If successful, these strategies would still allow maximal lean tissue growth and maximal feed efficiency but also result in carcasses with optimal quality.
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Footnotes
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1 Contribution No. 08-33-J of the Kansas Agric. Exp. Stn., Manhattan. 
2 Presented at the Growth and Development symposium at the annual meeting of the American Society of Animal Science, San Anto-nio, TX, July 8 to 12, 2007.
3 Corresponding author: bjohnson{at}ksu.edu
Received for publication July 24, 2007.
Accepted for publication October 23, 2007.
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Abstract
INTRODUCTION
