Robert A. Merkel Ronald E. Allen, Kenneth C ...

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Ronald E. Allen, Kenneth C. Masak, Pamela K. McAllister and. Concentration on Actin Synthesis in Cultured Satellite Cells. Effect of Growth Hormone, ...
Effect of Growth Hormone, Testosterone and Serum Concentration on Actin Synthesis in Cultured Satellite Cells Ronald E. Allen, Kenneth C. Masak, Pamela K. McAllister and Robert A. Merkel J ANIM SCI 1983, 56:833-837.

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EFFECT OF GROWTH HORMONE, TESTOSTERONE AND SERUM CONCENTRATION ON ACTIN SYNTHESIS IN CULTURED SATELLITE CELLS 1 Ronald E. Allen 2 , Kenneth C. Masak 3, Pamela K. McAllister 3 and Robert A. Merkel 3 University of Arizona, Tucson 85721 and Michigan State University, East Lansing 48824

interact directly with muscle to stimulate protein accretion, because neither growth hormone nor testosterone acted directly on muscle cells in vitro to stimulate muscle protein accumulation. (Key Words: Satellite Cell, Growth Hormone, Testosterone, ~-Actin, Muscle Cell Culture, Muscle Growth.)

Summary

Because most of the DNA in a m a t u r e muscle accumulates during postnatal life and is derived from satellite cells, cellular regulation of protein accumulation in muscle fibers originating from satellite cells is an important aspect of muscle growth control. These experiments were designed to study the modulation of a ot-actin accumulation in satellite cell-derived myotubes by serum and two anabolic hormones commonly assumed to be involved in muscle growth regulation; growth hormone and testosterone. Satellite cells were cultured from rats ranging in age from 5 d to 1 yr. After fusion into myotubes, various levels of pig serum, 3 to 20%, were added to culture medium, and the amount of c~-actin per m y o t u b e nucleus was determined 4 d later. The quantity increased with increasing percentages of serum in the medium. For an assessment of the extent to which serum stimulatory activity was due to growth hormone or testosterone in the serum, similar experiments were conducted with medium Containing 10% pig serum plus various levels of porcine growth hormone or testosterone. Neither of these hormones stimulated actin accumulation at concentrations from 10 -9 to 10 -6 M, a range encompassing physiological levels for each hormone. These experiments do not support the premise that either growth hormone and(or) testosterone are the blood-borne agents that

I ntroduction

Accumulation of DNA in skeletal muscle during postnatal growth has been extensively documented (Enesco and Puddy, 1964; Moss et al., 1964; Cheek et al., 1965; Harbison et al., 1976; Johns and Bergen, 1976) and has been shown to contribute most of the DNA in a mature muscle. The origin of this DNA has also been described and has been attributed to the process of satellite cell proliferation and differentiation (reviewed by Allen et al., 1979). Satellite cells are small, mononucleated myogenic cells that reside between the sarcolemma and the basement membrane of skeletal muscle fibers. Upon receiving the appropriate stimulation, as y e t unknown, these ceils proliferate and one or both daughter cells differentiate and attain the capacity to make molecules necessary for fusion with adjacent fibers. The nucleus from the satellite cell then becomes one of many muscle fiber nuclei and, as such, becomes involved in directing the synthesis of muscle proteins. Because k n o w l e d g e of t h e regulation of muscle protein accretion is critical to our understanding of meat animal growth, experiments were conducted to elucidate some of the factors that impinge directly on muscle ceils to stimulate the accumulation of muscle protein. For these in vitro experiments, cultured muscle

Michigan Agr. Exp. Sta. Article No. 9930 and Arizona Agr. Exp. Sta. Journal Paper No. 3421. This work was supported by Michigan Agr. Exp. Sta. Project No. 1280 and 1182 and a grant from the USPHS R23 AGO1467. 2Dept. of Anim. Sci., Univ. of Arizona. 3Dept. of Anim. Sci., Michigan State Univ. 833

JOURNAL OF ANIMAL SCIENCE, Vol. 56, No. 4, 1983

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fibers derived from satellite cells, the source of most muscle nuclei, were employed. Initial experiments were designed to examine the responsiveness of cultured muscle fibers to serum and to growth hormone and testostero n e - t w o hormones generally considered to have anabolic effects. Materials and Methods

Cell culture medium, pig serum and antibiotics were purchased from Grand Island Biological Co. 4 Acrylamide, ampholytes and stain for isoelectric focusing were purchased from Bio-Rad Laboratories s . Testosterone was purchased from the Sigma Chemical Co. 6, and growth hormone (3 IU/mg) was a gift from Dr. L. J. Machlin. Male Long-Evans rats from the following four age groups were used in these experiments: neonates (1 wk), rapidly growing rats (1 to 3 too), adult rats (9 to 12 mo) and old rats (24 mo). Within a given experiment skeletal muscle from three to five rats was pooled, and satellite cells were cultured according to the procedure of Allen et al. (1980b). Briefly, this procedure entails the liberation of satellite cells from minced muscle by digestion with pronase, followed by separation from fiber fragments by differential centrifugation. Cells were subsequently preplated for 24 h in dishes that had not been coated with collagen. During this preplating period, many of the contaminating fibroblasts attached to the surface of the dish, leaving a satellite cell-enriched population of cells in suspension. Cells remaining in suspension were plated in collagen coated tissue culture dishes in the presence of Eagle's minimum essential medium, 10% pig serum and an antibiotic mixture containing penicillin, streptomycin and gentamycin. Cells remained in this medium for approximately 3 d, until the major burst of cell fusion began. Cells were subsequently washed with buffered saline solution (BSS) and refed the various treatment media

4 Grand Island, NY. s Richmond, CA. St. Louis, MO. Testosterone assays were performed by Dr. G. D. Riegle, Dept. of Physiology, Michigan State Univ., East Lansing, MI. SGrowth hormone assays were performed by Dr. R. W. Hammond and M. J. DeGeeter, Monsanto Co., St. Louis, MO.

containing different concentrations of hormones or serum. Treatment media were replaced daily. After 4 d of exposure to treatment media, the experiments were terminated, and cultures were harvested for actin analysis or fixed and stained with Giemsa for determination of the number of nuclei in fused cells per culture dish. The same lot of serum was used in all experiments to eliminate minor variations in growth promoting activity. The serum was prepared by pooling blood from many animals at the time of slaughter. Because this serum was derived from market-age gilts and barrows, the testosterone level was very low (.24 ng/ml) 7. Furthermore, pooling the blood from many animals should reduce the impact of episodic release of hormones, such as growth hormone. Growth hormone concentrations in pig serum from two separate lots obtained in this manner were 4.1 and 4.9 ng/mlS; this is in general agreement with average growth hormone levels in pigs of this age as reported by Althen and Gerrits (1976). Because serum constituted only 10% of the medium used in growth hormone and the testosterone experiments, the basal levels of endogenous hormones in the medium would be an order of magnitude lower than the previously mentioned concentrations. Additional growth hormone or testosterone was added to achieve concentrations ranging from 10 -9 M to 10 -6 M. The amount of ~-actin, the muscle-specific form of actin, was analyzed in a crude fraction of cytoskeletal and myofibrillar proteins. Initially, living cultures were placed in a cold room at 2 C for 30 min; this step removed some minor proteins that migrate dose to actin in isoelectric focusing gels and hence interfere with quantitation. After the 30 min period, culture medium was removed and replaced with 50 mM KCI, 10 mM Tris-HCl, .5 mM 2-mercaptoethanol, .5% Triton X-100, pH 7.6. Cells were scraped from the dish in this solution, and the insoluble residue was collected by centrifugation at 10,000 x g for 15 min. The actin-containing cell residue was resuspended in the same solution and centrifuged a second time under the same conditions. The pellet was resuspended in 50 mM NaC1 and subjected to centrifugation at 10,000 x g for 15 min. This step was repeated, and the pellet obtained after the last centrifugation step was prepared for isoelectric focusing according to the method of O'Farrell (1975). Samples were subjected to isoelectric focus-

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ACTIN SYNTHESISIN CULTUREDSATELLITECELLS ing, and the alpha isozyme of actin was quantitatively measured by densitometry as described by Allen et al. (1980a). The amount of 0l-actin per culture divided by the number of fused cell nuclei per culture provided a basis for comparing muscle specific protein accumulation among cultures of various treatment groups. An important experimental point should be emphasized. Virtually all primary muscle cell cultures contain fibroblasts. Furthermore, fibroblasts also make contractile proteins similar to skeletal muscle actin and myosin. Consequently, total protein determinations are of limited value in satellite cell cultures, because they measure protein accumulation in muscle cells as well as that in the contaminating fibroblasts. The degree to which fibroblast contamination varies and the degree to which fibroblasts and muscle cells respond differently to a given treatment introduces an inestimable uncertainty that severely limits interpretation of this kind of experiment. Theoretically, measurement of myosin accumulation, by traditional sodium dodecyl sulfate (SDS) gel electrophoresis techniques, also suffers from the same problem because of the presence of fibroblast myosin that is indistinguishable from skeletal muscle myosin. The problem is not as severe as when total protein is being measured, because fibroblasts accumulate only about 10% as much myosin per nucleus as do muscle fibers (Young et al., 1979). In experiments in which fibroblast contamination is great or highly variable, however, this problem may be significant. Because of these potential pitfalls in protein accumulation measurement, we chose to monitor the accumulation of ~-actin, a muscle specific protein not synthesized by fibroblasts or presumptive myoblasts. The alpha isozyme of actin can be easily separated from the beta and gamma forms by isoelectric focusing; therefore, quantitation of c~-actin in isoelectric focusing gels allows the analysis of a protein made exclusively by terminally differentiated muscle cells while eliminating the problem of fibroblast actin contribution to the protein analyses. Each experiment represents the cultures derived from a given preparation of satellite ceils. Treatments within experiments refer to the different concentrations of growth hormone or testosterone administered to cultures. Five to eight 35 mm satellite cell cultures comprised each treatment group. Each individual growth hormone and testosterone experiment was

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subjected to an analysis of variance. A total of 18 testosterone experiments were conducted, five experiments each with neonates and adults, six experiments with 1 to 3-mo-old rats and two experiments with old rats. Eleven growth hormone experiments were conducted, four each with neonates and 1 to 3-too-old rats and three experiments with adult rats. Old rats were not available for growth hormone experiments. Results and Discu=sion

If protein accumulation in muscle fibers is controlled by factors circulating in blood and if our muscle cell culture system was capable of monitoring this activity, satellite cell-derived muscle fibers should accumulate increasing amounts of muscle protein in response to increasing concentrations of serum in the cell culture medium. Results from one of the experiments conducted to investigate this hypothesis are presented in figure 1. When serum concentration in the medium increased from 3 to 15%, the amount of ~-actin per muscle fiber nucleus increased. Beyond 15%, however, ~-actin accumulation began to plateau, or decline slightly. These results confirmed the assumption that serum-borne factors can modulate protein accretion in muscle fibers, but more importantly, they established that this assay system was capable of detecting such modulations in muscle protein accumulation. Because satellite cells are very sparse in postnatal muscle, large quantities of muscle must be

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I 15 20 SERUM LEVEL (%) Figure 1. Accumulation of c~-actin in satellite cellderived myotubes in response to various levelsof serum in the culture medium.

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processed through a relatively expensive and laborious procedure to acquire enough cells to establish sufficient numbers of 35 mm culture dishes to conduct these experiments. This was a limiting factor in our ability to c o n d u c t these experiments. Our reasons for using 10% serum instead of a lower percentage stem from a need to acquire adequate quantities of actin for analysis. Lower serum concentrations yielded cultures with actin contents near the lower limits of our ability to accurately measure this protein. To increase the probability of getting useful actin determinations from a high percentage of our cultures, we opted to increase serum concentration. Clearly, there is a tradeoff in this situation. Having established that muscle fibers derived from satellite cells are responsive to serum-borne agents, we attempted to elucidate factors responsible for stimulating muscle protein accumulation. Initial experiments were designed to examine the possibility that either growth hormone or testosterone could be responsible for this activity. The experimental protocol was similar to that used for studying the effect of serum; however, only one level of serum was used, and various levels of hormone were added to the medium. The medium contained 10% pig serum, a concentration within the linear region of the dose-response curve, and 10 -9 M and 10 -6 M hormone. A typical dose-response experiment for testosterone in cultures of neonatal rat muscle satellite cells is shown in figure 2. No significant differences among hormone treatments were observed in this experiment, nor were there any consistent differences in experiments conducted with cells representing the other age groups, including the old rats. These results suggest that the anabolic influence of testosterone is exerted on some other aspect of muscle growth, such as proliferation of satellite cells, or through an intermediate growth factor. The observations of Powers and Florini (1975), regarding the stimulation of myogenic cell proliferation by testosterone, provide a basis for suggesting that testosterone may act by stimulating satellite cell proliferation. Growth hormone was also ineffective in promoting c~-actin accumulation in satellite cell-derived muscle fibers in experiments with cells from rats in any of the three age groups (figure 3). Failure of growth hormone to stimulate muscle protein accumulation in muscle fibers in vitro could have been predicted

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