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Hormonal Regulation of Growth F. C. LEUNG Animal growth is a complex physiological process regulates] by the endocrine system (Figure 1), which also mediates the effects of nutritional, environmental, and genetic factors in animals. To enhance growth and improve feed conversion efficiency in agri- cultural animals, scientists must understand the roles of hormones (pepticle and steroid) and peptide growth factors in these pro- cesses and identify the limiting factors so that these processes can be modulated. The hormones that affect growth in ani- mals are growth hormone, insulin, thyroid hormones, glucocorticoids, prolactin, and gonaclal steroids (androgens and estrogens). Their role in growth and development has traditionally been investigated by examining the effect of hormone deprivation after organ ablation; the effects of excess amounts of hormones can be observed by administering the hormones to animals in vivo. Growth hormone (GH) is generally be- lieved to be the most important hormone affecting growth and development. Clinical observations show that GH deficiency in children results in dwarfism ant] that excess GH results in acromegaly and gigantism (Underwood! and Van Wyk, 1981~. This has 135 led to the assumption that an increase in the circulating concentration of GH would result in faster growth. This hypothesis has been confirmed] by the gene insertion tech- nique. Palmiter et al. (1983) proclucec] trans- genic mice by direct injection of cloned rat GH or human GH recombinant DNA, li- gated with a mouse metallothionein pro- moter, into the pronuclei of fertilized eggs. Transgenic mice that carried the extra GH gene, and that therefore had high circulating concentrations of GH, grew to twice the size of their control littermates. Hammer et al. (1984) also user! this technique to correct dwarfism in a strain of"Little" mice, which are deficient in GH; the transgenic mice grew even larger than normal mice. Injected GH has been reported to im- prove the growth rate and feed conversion efficiency of normal pigs (Chung et al., 1985; Machlin, 1972), calves (Brumby, 1959), and lambs (Wagner and Veenhuizen, 1978~. Administration of GH to dairy cows report- edly increases the efficiency of milk pro- cluction (see the papers by Gorewit ant] Linn in this volume), and in pigs and lambs shifts carcass composition from fat toward protein ant] moisture (Chung et al., 1985;
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136 Effects ~ I Hypothalamus Growth Hormone Releasing Somatostatins Factor ~ 1 Indirect Effects , ~ , E] ~ I Other Organs I Skeletal: Chondrogenesis, Skeletal Growth Extraskeletal: DNA-, RNA-, Protein Synthesis; Cell Prollferatlon 1 ~ Direct Effects 1 A+ |Other Organs | Effects Glucose Transport Glucose Homeostasis Amino Acid Transport Upolysis RNA- Protein Synthesis (Ever) FIGURE 1 Regulation and effects of growth hor- mone. Machlin, 1972; Wagner and Veenhuizen, 1978~. The effects of exogenous GH on growth in fish (salmon and trout) and chick- ens have recently been reported by Kawau- chi et al. (1986) and Leung et al. (1986b). However, responses in these animals were much less marked than those observed in . . transgen~c mice. To investigate the impact of increased circulating GH concentration on growth and feed efficiency, Leung et al. (1986b) used various experimental approaches to manip- ulate the endocrine systems of chicks. A discussion of their methodologies and re- sults follows. THE INFLUENCE OF GlI ON GROWTH Pituitary GH synthesis and reaction are generally believed to be regulates! by the hypothalamic releasing factor, GH releasing factor (GRF) and inhibiting factor, and so- matotropin releasing/inhibiting factor. In avian species, a thirc] hypothalamic factor, thyrotropin releasing hormone (TRH), which stimulates thyrotropin stimulating hormone APPENDIX at the pituitary level, is also a potent GH releaser (Harvey et al., 1978~. In contrast to mammalian species, where there is only one GH releasing factor, avian species ap- pear to have two. It is widely thought that the lipolytic effect of GH is direct but that somatomedin-C (SM-C) mediates the growth response of GH (Chawla et al., 1983; Un- clerwood and Van Wyk, 1981~. There is also evidence that GH may act directly in the tibia to promote bone growth (Isaksson et al., 1982; Russell and Spencer, 1985~. The various experimental methods used to ele- vate serum concentrations of GH are listed in Table 1. Effects of Chicken GH on Body Weight Gain in Chickens Large quantities of chicken pituitary GH were purified to examine its eject on growth (Leung et al., 1986b). The purified chicken GH (cGH), which was biologically active in the rat tibia bioassay, gave a dose-dependent response parallel to that of the bovine GH stanclard. The amino acid composition of cGH was similar to that of mammalian GH, and particle-sequencing analysis of cGH shower! 79 percent homology with bovine GH. Four-week-old Hubbard x Hubbard broiler cockerels were user] in all experi- ments. Thirty-six bircis were individually caged in a temperature- and light-controlled TABLE 1 Methods for Elevating Serum Concentration of Growth Hormone I. Treat with GH. 2. Treat with GRF for TRH. 3. Increase secretion of endogenous GRF or TRH by control of neuroregulators. 4. Decrease secretion or action of endogenous so- matomedin releasing/inhibiting factor. 5. Increase secretion of endogenous GRF or GH by inserting multiple copies oftheir structural genes, linked to an appropriate promoter, into the chicken genome.
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HORMONAL REGULATION OF GROWTH room; they were randomly divided into four treatment groups of nine birds each, with food and water available ad libitum. The purified cGH was dissolved in phys- iological saline and given daily by intrave- nous injection via the brachial vein at concentrations of 5, 10, ant! 50 ~g/bird in 100- volumes. Body Sleight ant! feed con- sumption were recorded twice weekly for 2 weeks. At the end of the experiment, birds were killed, clefeathered, and ground in a meat grinder. Tissues were analyzer! by New Jersey Feet] Laboratory, Inc. (Plains- field, Nail.), for moisture, protein, ant! fat content, according to the procedure rec- ommenclec] by the Association of Official Analytical Chemists. Birds that received 5 log of cGH daily shower! significant weight gains (20.6 and 13.5 percent over control birds) on clays 3 and 6, respectively. Birds that received 10 fig of cGH also showed significant weight gains over control bircis after 3 and 6 days of treatment (19.6 and 11.3 percent, re- spectively). Birds that received 50 log of cGH shower! an improvement in weight gain over control bircis, but the increase was not statistically significant. Overall, the increase in body weight gain seemed to be transient, so that the stimulating effect of cGH was diminished by the end of the experiment. There was no difference in the effect of feed conversion efficiency on car- cass composition between cGH-treated and control birds. Effects of Human Pancreatic GRF and TRH on Body Weight Gain in Chickens Chicken hypothalamic GRF has not yet been isolated and purified, but a synthetic human pancreatic GRF (hpGRF) has been shown to be active in stimulating cGH release in chickens both in viva and in vitro (Leung ant! Taylor, 1983; Scanes et al., 1984~. In addition, TRH, which is a hypo- thalamic peptide, has been shown to stim 137 ulate cGH release in viva. The objective of the studies clescribed below was to deter- mine the effect of hypothalamic peptides on growth in chickens. Four-week-olc! Hubbard x Hubbard broiler cockerels were used in all experi- ments. In the hpGRF experiment, bircis were individually caged and randomly dis- tributec3 into four treatment groups of nine birds each. In the TRH experiment, bircis were individually cager! and randomly di- vided into four treatment groups of 8 to 10 birds. All birds were housed in a tempera- ture- ant! light-controllecl room (25°C; 14 hours of light, 10 hours of darkness) and provided with food and water ad libitum. Food consumption and weight were re- corded twice weekly for 2 weeks. At the end of the experiment, birds were killed and defeathered, and carcass composition was analyzed as described in the previous section. The hpGRF44 (Bachem, Torrance, Calif. ~ and TRH (Beckman, Palo Alto, Calif. ~ were dissolved in physiological saline and injected via the brachial vein at concentra- tions of 0. 1, 1. 0, or 10.0 Catbird in a 100- ~1 volume. Control birds received 100 Al of a saline solution. Birds that received 0.1 log of hpGRF daily showed a significant increase in body weight gain early on, but that soon diminished. The similarly transient stimulating effect of cGH and hpGRF on body weight gain suggests that hpGRF is also mediated through pituitary GH. Birds that received 1.0 or 10.0 log of TRH dally showed significant increases in body weight compared to controls. In contrast to the effect of hpGRF, the growth response to TRH injections was not transient (Leung et al., 1984c). The difference between the effects of the two hormones is probably due to the additional stimulation of thyroid hor- mone by TRH. Thyroid hormones (triio- dothyronine tT3] and thyroxine itch) have been shown to influence body weight gain in chickens (Leung et al., 1985~.
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138 Somatome~in-c The growth activity of GH is believed to be mediated by SM-C growth factor, gen- eratec] mainly in the liver. Somatomedin-C is GH-`lepenclent, and purified SM-C has been shown to stimulate body weight gain in both hypophysectomized and intact rats (Hizuka et al., 1986; SchoenIe et al., 1982~. Since chicken SM-C has not been isolated ant] purified, a human SM-C raclioimmu- noassay (RIA) was used to measure serum immunoreactive SM-C when purified cGH was injected into 4-week-old cockerels (Leung et al., 1986b). Purified cGH clid not affect weight or incorporation of 3H-proline or 35SO4 in 9- to 10-day-old chicken embryo cartilage cultured in vitro, but purified hu- man SM-C hac3 a significant effect (Burch et al., 1985~. Thus' it seems that the growth promotion axis of hypothalamic GRF-pitui- tary GH-hepatic SM-C in chickens is similar to that in mammals, but investigation of the biological effects of purifier! chicken GRF and chicken SM-C is neecled to validate this hypothesis. Growth Hormone Receptor Hormone-receptor interaction is the first step in hormone action, but receptor phys- iology has only recently been given atten- tion. Many human diseases are known to result from receptor clefects, but the bio- logical significance of the receptor is only beginning to be recognized. For example, analysis of the amino acid] and nucleoticle sequences of purified epidermal growth fac- tor receptor (EGF-R) has enabled scientists to link the structure-function relationships of oncogenes (v-erbB) ant! EGF-R (Down- warc! et al., 1984~. Although there is no structural analysis (amino acid response) for the GH receptor as yet, its eventual deter- mination will lead to an understanding of the molecular basis of GH action. Leung et al. (1984a) demonstrated a spe- cific hepatic GH receptor in chickens and obs erve cl paradoxically high blood co ncen APPENDIX "rations of GH, as measurer] by a homolo- gous cGH RIA (Leung et al., 1984b), in sex- linkecl dwarf chickens (Lilburn et al., 1986~. These chickens grew to less than half the size of normal chickens, leacling Leung et al. (1984a) to examine GH receptor binding in the same strain. There was a significant decrease in hepatic receptor binding at 6, 8, and 20 weeks of age compared to that of normal, fast-growing broiler chickens (Leung et al., 1987~. Huybrechts et al. (1985) re- portec] that sex-linkec3 dwarf chickens also hac! significantly lower circulating immu- noreactive SM-C concentrations compared to those of normal birds. And Leung et al. (1984a) observed that sex-linked dwarf chickens had significantly higher hepatic (IGF-I) receptor binding. These observations may provide evidence that dwarfism is sex-linked and may be clue to a defect in the GH receptor. Based on preliminary results, we believe that GH receptors may be the limiting factor in the growth promoter axis in chickens. For ex- ample, normal Leghorn chickens, which grow at a much slower rate than broiler chickens, possess significantly fewer GH receptors than broiler chickens (Leung et al., 1987~. However, that hypothesis floes not agree with data reported for mammalian species. Growth hormone has been shown to maintain its own receptors in rat a(lipo- cytes ant] to up-regulate its hepatic recep- tors (Baxter and Zaltsman, 1984~. Recently, Chung ant] Etherton (1986) reported that the number of hepatic GH receptors is increased in pigs that have received GH injections. The method of regulating GH receptors in other agricultural animals is not known. However, if GH up-regulates its receptors at the target tissue, it is logical to assume that an increase in circulating GH would! result in an amplified biological response to GH. Gene Insertion The technology for introducing foreign genes into mammalian embryos forms the
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HORMONAL REGULATION OF GROWTH basis of a powerful approach for studying gene regulation and the genetic basis of development (Palmiter and Brinster, 1985~. A dramatic growth increase in transgenic mice from eggs that were microinjectec] with a metallothionein GH foreign gene suggests that this technology could be val- uable for agricultural applications. Indeed, Hammer et al. (1985) successfully intro- duced foreign genes into the genes of rab- bits, sheep, and pigs by microinjecting eggs, using mouse metallothionein-human GH recombinant DNA. The foreign DNA was integrated ant] expressed in transgenic rab- bits and pigs. Thomas E. Wagner (Ohio University, personal communication, 1986) also successfully introduced foreign genes in pigs by microinjection. Leung and co- workers have attempted to directly inject foreign DNA into the blastoderm of freshly laid eggs with recombinant DNA technology (unpublished! data). And Souza et al. (1984) user] the retroviral approach in introducing foreign genes into chickens. Kopchick et al. (1985) constructed a re- combinant DNA (pbGH-4~-that is an avian retroviral long-terminal repeat (LTR), li- gatec! to the structural bovine GH (bGH) gene. This recombinant DNA is biologically active in a transient eukaryotic expression assay system. When this recombinant DNA was totally integrated into a mouse fibroblast cell line, mature bGH was expressed and secreted into the culture medium. Leung et al. (1986a) purified and characterized the recombinant bGH from culture medium and shower] that the recombinant bGH pos- sesses the same physiochemical and physical properties as native pituitary bGH. This recombinant bGH DNA was then intro- clucec3 into the germinal disk of the freshly lair! egg by opening a window in the egg and injecting various amounts of DNA in circular or linear form with a micropipette. Only seven of the chicks that hatched from the 3, 000 injected eggs had measurable circulating immunoreactive bGH. When serum samples were measured with both a homologous cGH RIA and a bGH RIA, the 139 cross-reactivity of purified cGH and bGH in the RIA was less than 5 percent. The expression of bGH was transient; no de- tectable immunoreactive bGH was present after 10 weeks of age. All the chickens were killed or crossed after sexual maturity. Tis- sue DNA was analyzed by dot blot and Southern gel assays. No measurable im- munoreactive bGH was detected by RIA from seven samples collected from first- generation offspring. It appears, therefore, that this method is inefficient. In addition, since the germinal disk in freshly laid eggs consists of at least 500 to 1,000 cells, even if the foreign DNA is integrated in the host cell genome it is unlikely that the foreign DNA will enter the germ line. Use of a retroviral vector to introduce foreign genes into chicken genes provides an alternative experimental approach. In- deed, Souza et al. (1984) generated a re- combinant retrovirus by cloning chicken GH cDNA into a modified Rous sarcoma virus Schmiedt-Ruspin A genome in which the sac gene was entirely deleted. Recom- binant infectious virus that expresses cGH was generated to infect 9-day-old chick embryos. Subsequently born chicks ex- pressed circulating concentrations of cGH that were two- to threefold higher than those of normal birds. In addition, the birds were uremic. Salter et al. (1986) obtained similar results using a different retroviral vector. These results suggest that the retro- viral approach may be more elective than direct injection of foreign DNA in intro- clucing foreign genes into the germ line of chickens. CONCLUSIONS AND FUTURE DIRECTIONS Our preliminary information that the GH receptor, rather than GH itself, may be the limiting factor in the growth production axis in chickens opens up new research direc- tions. Pituitary GH has been purified from many agricultural animals, and antibodies to these preparations have also been gen
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140 crated for RIA. Somatomedin-C has been purified only from humans and rodents (Spencer et al., 1983~; with recombinant DNA technology, scientists should be able to clone the SM-C gene and express syn- thetic recombinant SM-C using prokaryotic and eukaryotic cell expression systems. Only then can the biological activities of SM-C in agricultural animals be determined. The techniques for inserting foreign DNA into genes by microinjection into the pronucleus of fertilized eggs have been successful in agricultural animals (Hammer et al., 1986), and the retroviral vector approach in chick- ens is also promising. However, further research is needed to determine which genes are most desirable for use in gene insertion, define the sites of integration, and attain the fine control for expressing the exogenous genes that is necessary to make such technology useful to agriculture. ACKNOWLEDGMENTS I am grateful for the collaboration of Drs. John Kopchick, km Smith, H. Chen, and Mike Lilburn and for the expert assistance of I. Taylor, A. Van Iderstine, C. A. Ball, K. N. Ngiam-Rilling, B. Goggins, C. I. Rosenblum, R. Malavarca, E. Mills, and F. Macks. I also thank M. E. Mer~cka and H. B. Crow for typing this manuscript and D. L. FeIton for her expert editing. REFERENCES Baxter, R. C., and Z. Zaltsman. 1984. Induction of hepatic receptors for growth hormone (GH) and prolactin by GH infusion is sex dependent. Endo- crinology 115:2009. Brumby, P. J. 1959. The influence of growth hormone on growth in young cattle. N.Z. J. Agric. Res. 2:683. Burch, W. M., G. Corda, J. J. Kopchick, and F. C. Leung. 1985. Homologous and heterologous growth hormones fail to stimulate avian cartilage growth in vitro. J. Clin. Endocrinol. Metab. 60:747. Chawla, R. K., J. S. Parks, and D. Rudman. 1983. Structural variants of human growth hormone: Bio chemical, genetic and clinical respects. Annul Rev. Med. 34:519. Chung, C. S., and T. D. Etherton. 1986. Characteri APPENDIX zation of porcine growth hormone (pGH) binding to porcine liver microsomes: Chronic administration of pGH induces pGH binding. Endocrinology 119:780. Chung, C. S., T. D. Etherton, and J. P. Wiggins. 1985. Stimulation of swine growth by porcine growth hormone. J. Anim. Sci. 60:118. Downward, J., Y. Yarden, E. Mayes, G. Scarce, N. Totty, P. Stockwell, A. Ullrich, J. Schlessinger, and M. D. Waterfield. 1984. Close similarity of epider- mal growth factor receptor and v-erb-B oncogene protein sequences. Nature 307:521. Hammer, R. E., R. D. Palmiter, and R. L. Brinster. 1984. Partial correction of murine hereditary growth disorder by germ-like incorporation of a new gene. Nature 311:65. Hammer, R. E., V. G. Pursel, C. E. Rexroad, R. J. Wall, D. J. Bolt, K. M. Ebert, R. D. Palmiter, and R. L. Brinster. 1985. Production of transgenic rab- bits, sheep and pigs by microinjection. Nature 315:680. Hammer, R. E., V. G. Pursel, C. E. Rexroad, R. J. Wall, D. J. Bolt, R. D. Palmiter, and R. L. Brinster. 1986. Genetic engineering of mammalian embryos. J. Anim. Sci. 63:269. Harvey, S., C. G. Scanes, N. J. Bolton, and A. Chadwick. 1978. Effect of thyrotropin-releasing hor- mone (TRH) and somatostatin (GH-RIH) on growth hormone and prolactin secretion in vitro and in viva in the domestic fowl (Gallus domesticus). Neuroen- docrinology 26:249. Hizuka, N., K. Takano, K. Asakawa, M. Miyakawa, I. Tanaka, R. Harikawa, and K. Shizume. 1986. Insulin- like growth factor I stimulates growth in normal growing rats in viva. In Proceedings of the 68th Annual Endocrine Society Meeting, June 25-27, 1986, Anaheim, Calif. Bethesda, Md.: Endocrine Society. Huybrechts, L. M., D. B. King, T. J. Lauterio, J. Marsh, and C. G. Scanes. 1985. Plasma concentra- tions of somatomedin-C in hypophysectomized, dwarf and intact growing domestic fowl as determined bY heterologous radioimmunoassay. J. Endocrinol. 104:233. Isaksson, O. G. P., J.-O. Jansson, and I. A. M. Cause. 1982. Growth hormone stimulates longitudinal bone growth directly. Science 216:1237. Kawauchi, H., S. M. Ama, A. Yasuda, K. Yamaguchi, K. Shirahata, J. Kubota, and T. Hirano. 1986. Isolation and characterization of chum salmon growth hormone. Arch. Biochem. Biophys. 244:542. Kopchick, J. J., R. Malavarca, T. Livelli, and F. C. Leung. 1985. Use of avian retroviral-bovine growth hormone DNA recombinants to direct expression of bovine growth hormone by cultured fibroblasts. DNA 4:23. Leung, F. C., and J. E. Taylor. 1983. In viva and in vitro stimulation of growth hormone release in chick- ens by synthetic human pancreatic growth hormone releasing factor (hpGRF). Endocrinology 113:1913. , ,
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HORMONAL REGULATION OF GROWTH Leung, F. C., J. Gillett, M. S. Lilburn, and J. Kopchick. 1984a. Analysis of growth hormone re- ceptors and genes in sex-linked dwarf chickens. J. Steroid Biochem. 20:1557. Leung, F. C., J. E. Taylor, S. L. Steelman, C. D. Bennett, J. A. Rodkey, R. A. Long, R. Serio, R. M. Weppelman, and G. Olson. 1984b. Purification and properties of chicken growth hormone and the de- velopmentofahomologousradioimmunoassay. Comp. Endocrinol. 56:389. Leung, F. C., J. E. Taylor, and A. Van Iderstine. 1984c. Thyrotropin-releasing hormone stimulates body weight gain and increases thyroid hormones and growth hormone in plasma of cockerels. Endo- crinology 115:736. Leung, F. C., J. E. Taylor, and A. Van Iderstine. 1985. Effects of dietary thyroid hormones on growth, plasma T3, T4 and growth hormone in normal and hypothyroid chickens. Gen. Comp. Endocrinol. 59:91. Leung, F. C., B. Jones, S. L. Steelman, C. I. Rosen- blum, and J. J. Kopchick. 1986a. Purification and physiochemical properties of a recombinant bovine growth hormone produced by cultured murine fi- broblasts. Endocrinology 119:1489. Leung, F. C., J. E. Taylor, S. Wien, and A. Van Iderstine. 1986b. Purified chicken growth hormone (cGH) and a human pancreatic growth hormone releasing factor (hpGRF) increased body weight gain in chickens. Endocrinology 118:1961. Leung, F. C., W. J. Styles, C. R. Rosenblum, M. S. Lilburn, and J. A. Marsh. 1987. Diminished hepatic growth hormone receptor bindings in sex-linked dwarf broiler and Leghorn chickens. Proc. Soc. Exp. Biol. Med. 184:234. Lilburn, M. S., K. N. Ngiam-Rilling, J. H. Smith, and F. C. Leung. 1986. The relationship between age and circulating concentrations of triiodothyronine (T3), thyroxine (T4), and growth hormone in com- mercial meat strain chickens. Proc. Soc. Exp. Biol. Med. 182:336. Machlin, L. J. 1972. Effect of porcine growth hormone on growth and carcass composition of the pig. J. Anim. Sci. 35:794. Palmiter, R. D., and R. L. Brinster. 1985. Transgenic mice. Cell 41:343. 141 Palmiter, R. D., G. Norstedt, R. E. Gelines, R. E. Hammer, and R L. Brinster. 1983. Metallothionein- human GH fusion genes stimulated growth of mice. Science 222:809. Russell, S. M., and E. M. Spencer. 1985. Local injections of human or rat growth hormone or of purified human somatomedin-C stimulate unilateral tibial epiphyseal growth in hypophysectomized rats. Endocrinology 116:2563. Salter, D. W., E. J. Smith, S. H. Hughes, S. E. Wright, A. M. Fadly, R. L. Witter, and L. B. Crittenden. 1986. Gene insertion into the chicken germ line by retroviruses. Poultry Sci. 65:1445. Scanes, C. G., R. V. Carsia, T. J. Lauterio, L. Huybrechts, J. Rivier, and W. Vale. 1984. Syn- thetic human pancreatic growth hormone releasing factor (GRF) stimulates growth hormone secretion in the domestic fowl (Gallus domesticus). Life Sci. 34:1127. Schoenle, E., J. Zapf, R. E. Humbel, and E. R. Froesch. 1982. Insulin-like growth factor I stim- ulates growth in hypophysectomized rats. Nature 296:252. Sonza, L. M., T. C. Boone, D. Murdock, K. Langley, J. Wypych, D. Fenton, S. Johnson, P. H. Lai, R. Everette, R. Y. Hsu, and R. Bosselman. 1984. Application of recombinant DNA technologies to studies on chicken growth hormone. Exp. Zool. 232:465. Spencer, E. M., M. Ross, and B. Smith. 1983. The identity of human insulin-like growth factors I and II with somatomedins C and A and homology with rat IGF I and II. Proceedings of a Symposium on Insulin-Like Growth Factors/Somatomedins, Nai- robi, Kenya, November 13-15, 1982. Berlin: Walter de Gruyter. Underwood, L. E., and J. J. Van Wyk. 1981. Hormones in normal and aberrant growth. P. 1149 in Textbook of Endocrinology, R. H. Williams, ed. Philadelphia: W. B. Saunders. Wagner, J. F., and E. L. Veenhuizen. 1978. Growth performance, carcass deposition and plasma hor- mone levels in wether lambs when treated with growth hormone and thyroprotein. J. Anim. Sci. 45:397.
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Muscle Cell Growth and Development RONALD E. ALLEN Skeletal muscle from domestic animals is a major source of high-quality protein in the human diet. Past technological advances in production of animal muscle protein have been baser] on empirical and fundamental biological research. Future technological advances, however, are less likely to occur unless research is firmly grounder! in the basic biology of muscle and animal growth. The primary function of this paper is to review information about the structure and composition of muscle, muscle clifferentia- tion and development, and key elements of protein metabolism as they relate to muscle growth. It also describes current areas of active research interest and speculates on applications of new research knowledge and future research needs. MUSCLE CELL STRUCTURE AND COMPOSITION The differentiated muscle cell in postnatal muscle is the muscle fiber, a highly spe- cializecI, long, cylindrical cell that can range in diameter from 10 to 100 Em en c! in length from millimeters up to many centimeters. The primary differences in fibers of different 142 species are fiber length and number of fibers per muscle. Each fiber is surrounded by a 7.5- to 10-nm-thick plasmalemma, caller! the sarcolemma. The sarcolemma is a lipid bilayer like the cell membranes of other cells and has a lipid composition of roughly 60 percent protein, 20 percent phospho- lipid, and 20 percent cholesterol. Surround- ing the sarcolemma is the basal lamina, or basement membrane. This somewhat amor- phous structure, 50 to 70 nm thick, is composed of mucopolysaccharicles an(1 col- lagen (types III ant] V). The cell membrane of muscle has a specialized structure the motor endplatc which accommodates in- teraction with an axon from a motoneuron. In addition, the membrane maintains an electrical potential that is propagated from the motor en(lplate, clown the membrane, ant] finally into the cell by a complex set of invaginations that form the transverse tu- bular system. Muscle fibers contain the major orga- nelles present in most cells. The most strik- ing difference between muscle cells an(1 the majority of other cells is their multinu- cleated nature. Depending on its size, an in(lividual fiber may contain hundreds of
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MUSCLE CELL GROWTH AND DEVELOPMENT nuclei. They are found just beneath the sarcolemma and seem to be randomly dis- tributed along the length of the fiber. Mi- tochondria are present between the con- tractile elements of muscle; their concentration varies with the metabolic ac- tivity of the particular fiber. Ribosomes are dispersed within the cytoplasm, but very few are associated with endoplasmic retic- ulum, primarily because muscle fibers syn- thesize few secreted proteins. The enclo- plasmic reticulum in muscle has formed a specialized set of membrane structures called the sarcoplasmic reticulum. The primary function of this structure is regulation of free calcium ion concentration. When free calcium ion concentration is maintainer] be- low approximately 0.1 ~M, contraction does not occur. But when the membrane is depolarized, the action potential reaches the interior of the cell through the trans- verse tubular system, calcium is released from the sarcoplasmic reticulum, the con- centration approaches 1 ,uM, and contrac- tion is activated. Lysosomes are not readily seen in muscle fibers, although lysosomal enzymes are present. The lysosomes are most likely sequestered in the sarcoplasmic reticulum. By far the most unique subcellular aspect of muscle fibers is the contractile machinery, the myofibril. This is an aggregation of 12 to 14 proteins into highly organized con- tractile threads that are insoluble at the ionic strength of the cytoplasm in muscle cells. It is noteworthy that this specialized set of proteins constitutes about 55 percent of the total protein in muscle. Conse- quently, many developmental studies of muscle have focuses] on myofibrillar protein gene expression and synthesis, which are discussed later in this paper. Myofibrils are composed of two main classes of filaments: thick filaments and thin filaments. Thick filaments measure approx- imately 15 rim by 1,500 nm. The major protein in thick filaments is myosin, which has the active site that hydrolyzes adenosine 143 triphosphate (ATP) and the site that binds to actin in the thin filament. The thin filament is roughly 6 nm by 1,000 nm and is composed of actin, which forms the beaded backbone of the filament, ant! tropomyosin and troponin, which perform regulatory functions. At one end, thin filaments insert into a protein lattice called the Z-line; at the other end, they overlay with thick filaments in a hexagonal array. Aciclitional small-diameter filament systems are present within myofibrils to provide an elastic com- ponent. Also, an intermecliate-diameter fil- ament system, found outside the periphery of the myofibril, links adjacent myofibrils and maintains their contractile units in reg- ister. Specific details of the ultrastructure of myofibrils and the biochemical properties of this intercligitating array of filaments can be found in Goll et al. (1984~. These features of muscle cells are com- mon to all skeletal muscle fibers, but specific fibers have cli~erentiated somewhat de- pending on their purpose. Some populations of fibers are primarily responsible for rapid contractions on an intermittent basis, while others have slower contraction speed ant! sustain contractile activity over extended periods of time. Muscle fiber types have been described extensively in many species; and their biochemical, physiological, and morphological cli~erences are significant to problems of muscle growth and meat qual- ity. A generalized scheme for describing fiber types classifies them on the basis of their contraction speed and on the energy metabolism pathways primarily used to pro- vide energy for contraction. Peter et al. (1972) provided one of the most descriptive classification systems by grouping fibers into three general categories. Fibers that were dependent on oxiclative metabolism and had slower contraction speecis were classified as slow-twitch, oxidative fibers (SO). Fibers with faster contraction times that were de- pendent on anaerobic, or glycolytic, energy metabolism pathways were termed fast- twitch, glycolytic fibers (FG). A third broac!
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144 category contained fast-twitch fibers that had glycolytic metabolic capabilities but also a significant capacity for oxidative metabo- lism; these were termed] fast-twitch, oxida- tive-glycolytic fibers (FO G). Contraction speed is correlated with myosin aclenosine triphosphatase (ATPase) activity and, therefore, with the particular myosin isozymes synthesizer! by the fiber. Other myofibriliar protein isoform varia- tions may also be associated with contractile properties. The complexity and degree of development of the sarcoplasmic reticulum, t-tubule system, and neuromuscular junc- tions have all been associated with contrac- tion speed and fiber class. As expected, mitochondrial content and glycolytic en- zyme content vary among fiber types, as (lo energy substrates such as glycogen and triglyceride. Aspects of fiber type variation that affect muscle growth include the not- able differences in fiber size that generally correlate with muscle fiber type. SO fibers are smaller in diameter than FG fibers, and FO G fibers tent] to be intermediate in size. Smaller fiber diameters may facilitate effi- cient gas exchange in oxidative fibers. In addition, SO fibers tend to have higher nuclei concentrations and, therefore, lower protein concentrations per nucleus. Satellite cell frequency, however, is reportedly higher for SO fibers (Kelly, 1978b). Because indi- vidual muscles vary in fiber type composi- tion, factors that clifferentially affect the development or growth of specific fiber types can result in alterations in muscle mass (for example, the transition from FG to FO G that can accompany aerobic con- ditioning). Reductions in fiber diameter and, consequently, muscle mass would be ex- pected. Alterations in gene expression and in quantitative aspects of protein metabo- lism that are responsible for such fiber type transitions are poorly understood. Chemical composition of muscle tissue can be quite variable, and the primary source of variation is intramuscular adipose tissue. It is clear that most of the variation APPENDIX in major constituents is minimized when expressed on a fat-free basis. Some com- positional variation can be found in associ- ation with aging, but, in general, it is attributable to changes in moisture content. Skeletal muscle from very young animals has a high moisture content that decreases with maturity. As a result, protein concen- tration increases with maturity. Subtle changes in other constituents, such as gly- cogen, can vary among muscles and species, but these differences may not have major nutritional significance when considering the composition of muscle as a food. The primary lipid fraction contributing to muscle tissue variation is triglyceride, which is stored in adipocytes within the muscle. These depositions are commonly referred to as marbling, and within the range of marbling found in the longissimus muscle of beef, the ether-extractable lipid (primar- ily triglycericle) varies from 1.77 to 10.42 percent on a wet weight basis (Savell et al., 1986~. Cholesterol content, on the other hand, is less variable. This can best be understood in light of its role in muscle tissue. Choles- tero} is an integral part of cell membranes, mainly the plasma membrane. On a tissue basis across maturity groups and marbling contents within maturity groups, cholesterol content of beef muscle floes not vary (Stromer et al., 1966~. In addition, the amount of cholesterol per gram of whole steak was not significantly different among the five yield gracles examined by Rhee et al. (1982~. Furthermore, neither breed type nor nu- tritional background affected cholesterol content of lean muscle tissue in beef cows (Eichhorn et al., 1986~. It is possible to find variation in cholesterol content of meat, however, because adipose tissue tends to have a higher cholesterol concentration than do muscle fibers. Consequently, variations in the amount of subcutaneous or inter- muscular fat consumed with the lean portion can alter cholesterol intake. It has been calculated that 37 to 56 percent of the
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MUSCLE CELL GROWTH AND DEVELOPMENT cholesterol in a cooker] rib steak of beef originates from subcutaneous and inter- muscular adipose tissue (Rhee et al., 1982~. In looking only at muscle cells, however, significant variations in cholesterol content have not been seen, even among most of the species used for muscle foods (Reiser, 1975; Watt and Merrill, 1963~. This is also true for the amino acid composition of muscle. The majority of muscle cell proteins are myofibrillar ant! are very highly con- served across species. In addressing topics such as alteration of tissue composition to enhance nutritional quality, it is important to keep in mind that the biology of the animal or tissue must come first. Our ability to manipulate cells in animals has both physiological limits en c] ramifications. MUSCLE FIBER DEVELOPMENT Prenatal Development Myogenesis originates in cells of the em- bryonic mesoderm and apparently follows a similar course in all species examined. Per- haps the most detailed] descriptions come from studies of human (Hauschka, 1974) and chick (White et al., 1975) embryo clevel- opment. In the human, no apparent orga- nization is noted in the limb mesoderm on day 28 of development, but by day 43 loose connective tissue cell regions and compact myogenic cell regions are visible. By clay 45 the first small multinucleated myotubes (the precursors of muscle fibers) have formed; by day 50 the general organization of major muscles and bones is essentially complete. Beyond this point, the rate of muscle his- togenesis occurs at different rates between ant] within individual muscles. In the gas- trocnemius on day 62, well-developed, my- ofibril-containing muscle fibers are present, but the majority of cells are still mononu- cleated. This population decreases to about 50 percent of the total by clay 72, while fibers increase two- to threefolcl. During the next 2 weeks, fiber formation proceeds 145 rapidly, with the percentage of mononu- cleated cells diminishing to 20 percent by clay 95 and further decreasing to the point that only a few single cells persist in asso- ciation with fibers by day 146. In other vertebrate species, comparable developmental patterns are discernible. One striking observation in rat and chick muscle is the development of two populations of fibers (Kelly and Zacks, 1969; McLennan, 1983~. The "primary fibers" develop early and are surrounded by closely associated mononucleatec] cells. In the chick embryo, "seconclary fiber" formation proceeds rap- idly after about 12 days of development until most of the mononucleated cell pop- ulation is exhausted and fiber formation is complete. This occurs before hatching in the chick and before birth in most mammals. A similar biphasic developmental pattern has been documentecl in fetal lamb skeletal muscle (Ashmore et al., 1972~. In general, fiber formation is complete near the time of birth. The stucly of myogenesis focuses on the muscle development process and has cen- tered around efforts to unravel myogenic lineages and the mechanisms responsible for alterations in the synthetic programs of muscle cells that lead to the formation of fibers and the expression of muscle-specific cell characteristics. One of the most impor- tant initial observations on the mechanisms of myogenesis came from a series of exper- iments reported by Stockdale and Holtzer (1961) that directly (demonstrate that mul- tinucleated myotubes arise from the fusion of mononucleated myogenic cells (myo- blasts). Furthermore, only mononucleatec] cells have the ability to proliferate; the nuclei in myotubes cannot replicate their DNA and divide. Consequently, the tran- sition from a proliferating myoblast to a nonproliferating myotube that can synthe- size muscle-specific macromolecules rep- resents the terminal step in muscle differ- entiation. There now appear to be several different
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The Use of Bioassays To Detect and Isolate Protein or Peptide Factors Regulating Muscle Growth in Meat-Producing Animals WILLIAM R. DAYTON PEPTIDE FACTORS AFFECTING MUSCLE GROWTH Several peptide or protein factors that have the potential to regulate muscle growth in meat-producing animals have been iclen- tified. These are discussed below. Somatotropin The effect of somatotropin deficiency on muscle growth has been well established for many years. Long-term administration of somatotropin to pituitary-intact animals has been reporter] to increase muscling, decrease fat content, ant] improve feed efficiency in swine (Chung et al., 1985; Machlin, 1972~; increase nitrogen retention in steers (Moseley et al., 1982) and sheep (Davis et al., 1969~; increase growth rate in lambs (Wagner ant] Veenhuien, 1978~; and improve milk production in dairy cattle (Peel et al., 1981~. However, it appears unlikely that somatotropin directly affects proliferation and protein turnover in muscle cells. Although there is an increased incor- poration of 3H-thymicline into DNA in mus- cle from somatotropin-treated hypophysec tomizec] rats as compared to untreated controls (Breuer, 1969), this may reflect a direct effect of somatotropin on proliferation of nonmuscle cells or an indirect effect of somatotropin on proliferation of muscle cells. It has also been reported that in in vitro incubations of rat diaphragm muscle, 10-8M somatotropin stimulates amino acid uptake (Albertsson-Wikland and Isaksson, 19761. However, recent observations that many types of cells can secrete somatomedin (Ad- ams et al., 1984; Hill et al., 1986a) raise the possibility that responses seen in the intact diaphragm are the result of locally produced somatomedins. In fact, it is generally be- lieved that many if not all of the effects of somatotropin on muscle growth are me- diated through somatotropin-clependent plasma factors somatomedins produced in response to somatotropin. In culture, muscle cells do not appear to respond! to the addition of physiological levels of somatotropin. Ewton and Florini (1980) have reported that somatotropin has no detectable effect on anabolic processes in embryonic muscle cell cultures. Ad(li- tionally, Allen et al. (1983) have reported that somatotropin has no direct effect on 173
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174 the rate of actin synthesis in myotube cul- tures derived from rat satellite cells. These findings support the theory that the eject of somatotropin on muscle is an indirect one mediated through the somatomedins. Insulin-Like Growth Factors (Somatomedins) Insulin-like growth factors are small poly- peptides (approximate molecular weight of 7,500 gallons) extracted and purified from human serum. They possess insulin-like properties in vitro but do not cross-react with insulin antibodies. Multiplication stim- ulating activity (MSA) is the name given to a family of polypeptides isolated from media conditioner! by a Buffalo rat liver (BRL) cell line (BRL 3A). To date, two classes of insulin-like growth factors (IGFs) have been characterized: IGF-I, also referrer] to as basic somatomedin (pH 8.~8.4), or soma- tomeclin-C (SM-C), and IGF-II, or neutral somatomedin. Multiplication stimulating activity appears to be the rat form of IGF- II, since the primary structure of M SA shows 93 percent identity with that of hu- man IGF-II (Marquarcit et al., 19814. At concentrations of 10-9 to 10-~°M, IGFs are mitogenic for a variety of cultured cell types. Biologically active receptors for both IGF- I/SM-C ant] IGF-II/MSA have been iden- tified on the surface of cultured muscle cells (Ballard et al., 1986~. IGF-I/SM-C has been shown to stimulate growth of hypophysec- tomized rats (Schoenle et al., 1982), prolif- eration of cultured myoblasts (Ballard et al., 1986), amino acid uptake in cultured myob- lasts (Hill et al., 1986a), differentiation of cultured myoblasts (Ewton and Florini, 1981), and RNA synthesis and polypepticle chain initiation in an isolated muscle (Monier and Le Marchand-Brustel, 1984~. IGF-II/MSA has been shown to stimulate proliferation of cultured myoblasts (Ewton and Florini, 1981; Florini and Ewton, 1981; Florini et al., 1984), amino acid transport into cultured muscle cells (Janeczko and Etlinger, 1984), APPENDIX and the rate of protein synthesis in cultured myotubes (.Janeczko and Etlinger, 1984~. MSA has also been shown to decrease the rate of protein degradation in cultured my- otubes (Janeczko ant] Etlinger, 19844. In addition to their well-documented presence in serum, both IGF-I/SM-C and IGF-II/ MSA have been reported to be released by rat myoblasts (Hill et al., 1986b), thus raising the possibility that these peptides may be involved in autocrine or paracrine regulation of muscle growth. On the basis of this information, it appears likely that insulin- like growth factors are potent stimulators of all aspects of muscle growth and clevelop- ment. Insulin The role of insulin in regulating general cell metabolism has been recognized for many years, but its mechanism of action is still not well understood. Similarly, its role in controlling muscle growth is not clear. Several lines of evidence suggest that insulin may have an anabolic effect on muscle tissue. Studies of a variety of animal models have demonstrated that wasting of skeletal muscle is a prominent feature of diabetes mellitus and that it is reversed by admin- istration of insulin (Pain and Garlick, 1974~. Aclclitionally, ribosomes isolated from mus- cle of diabetic rats are less active in in vitro protein synthesis systems than in ribosomes from nondiabetic controls. In vitro stu(lies with isolates! muscles (Fulks et al., 1975) and the perfused rat hemicorpus Ue~erson et al., 1977) have shown that insulin in- creases the rate of protein synthesis and decreases the rate of protein degradation in these systems. In cultured muscle cells as well as in fibroblasts and fibroblastic cell lines, supra- physiological concentrations of insulin (21 ,u~g/ml) are required to elicit a maximum response. In muscle cell cultures, these high concentrations stimulate both prolif- eration and differentiation of myogenic cells
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BIOASSAYS (Ewton ant] Florini, 1981~. Insulin at high concentrations (10-6M) is a component of synthetic media used to support growth and differentiation of myogenic cells in culture (Dollenmeier et al., 1981; Florini and Rob- erts, 1979~. It has been proposed that the stimulation of growth of fibroblasts by in- sulin is mediated by insulin's weak binding to receptors for insulin-like growth factors. Affinity cross-linking studies have shown the existence of two classes of IGF recep- tors. Type I receptors (Massague and Czech, 1982) have a higher affinity for IGF-I than for IGF-II and a low affinity for insulin. The structure and subunit composition of type I receptors are very similar to those of the insulin receptor. Type II receptors bind IGF-II with a higher affinity than they do IGF-I and do not appear to have appreciable affinity for insulin (Massague ant! Czech, 1982~. At high concentrations, insulin may bind to the type I receptor, and in so doing affect cell growth in a manner similar to that observed for much lower concentrations of IGF-I. This hypothesis is based on work by King et al. (1980), who showed that blockade of high-affinity insulin receptors with anti-receptor Fab fragments blocked high-affiinity insulin binding but did not prevent insulin-induced stimulation of DNA synthesis in cultured fibroblasts. Further- more, these investigators showed that anti- insulin-receptor immunogIobulin G (IgG), which triggers a number of acute insulin- like metabolic effects, floes not stimulate DNA synthesis. They concluded that the growth-promoting effects of insulin on hu- man fibroblast were clue to binding of insulin to the type I receptor. Although this has not been prover! in cultured muscle cells, it would seem likely that the well-clocu- mented effects of supraphysiological con- centrations of insulin on proliferation and differentiation of cultures] muscle cells are the result of this spillover action of insulin through IGF-I receptors. Insulin has a wide range of effects on cell metabolism. Consequently, it is possible 175 that physiological levels of insulin facilitate muscle cell growth by maintaining cells in a metabolic state that allows them to respond] to other hormones and growth factors that stimulate cell proliferation. Differentiation Inhibitor Coon's BRL cells secrete a protein that is a potent inhibitor of skeletal myoblast differentiation in vitro (Evinger-Hodges et al., 1982; Florini et al., 1984~. In skeletal myoblast cultures, this protein reversibly blocks fusion, elevates creatine kinase, and increases binding of alpha-bungarotoxin. It has also been isolates] from sera of embry- onic origin, prompting the suggestion that it may play a role in embryonic growth of myoblasts and in satellite cell formation (Evinger-Hociges et al., 1982~. Transferrin Tr ransterr~n Is an iron-bin(ling glycopro- tein that is present in serum (Ozawa and Kohama, 1978) and embryo extract (Ii et al., 1981~. Additionally, transferrin-like mol- ecules have been isolated from both nerve and muscle extracts (Matsuda et al., 1984~. In muscle cell cultures, iron-saturated trans- ferrin stimulates both proliferation and dif- ferentiation and is essential for maintenance of healthy myotubes. The effect of transfer- rin on muscle growth in culture is absolutely dependent on the presence of iron and appears to be class specific (that is, mam- malian transferring do not affect avian myo- blasts, nor do avian transferring affect mam- malian myoblasts) (Shimo-Oka et al., 1986~. Fibroblast Growth Factor In cell cultures, fibroblast growth factor (FGF) stimulates proliferation of myogenic cells and delays their clifferentiation (Gos- podarowicz et al., 1976; Linkhart et al., 1981~. Allen et al. (1984) have proposed that FGF regulates satellite cell proliferation in
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176 skeletal muscle. However, they do not be- lieve that serum is the source of the FGF that is affecting satellite cell proliferation. Rather, they hypothesize that FGF-like molecules are producer! locally in muscle and trigger a localizes! response of satellite cells cluring muscle regeneration. Paracrine and Autocrine Control of Muscle Growth Reports that various cell types secrete growth factors have sparked interest in au- tocrine and paracrine regulation of muscle growth. It has been reported that cultured fibroblasts secrete IGF or IGF-like mole- cules (Adams et al., 1984) ant] that fetal rat chondrocytes sequentially elaborate sepa- rate growth- ant! clifferentiation-promoting peptides cluring their development (Shen et al., 1985~. Aclditionally, cultured myo- blasts have been reported to synthesize and secrete IGF-I/SM-C (Hill et al., 1986a). Because all these cell types are fount! in muscle tissue, their ability to produce growth factors raises the possibility that muscle growth may be at least partially regulated by factors procluced locally. This hypothesis is supported by reports of the purification of an FGF-like muscle growth factor present in skeletal muscle tissue (Karkami et al., 1985~. The mechanism by which this factor is accumulated in skeletal muscle and the relationship of this accumulation to regula- tion of muscle growth and regeneration is of interest. BIOASSAYS FOR FACTORS INFLUENCING MUSCLE GROWTH To develop effective strategies for con- trolling animal growth, a better understand- ing is needed of the mechanism by which known growth factors regulate proliferation, (differentiation, and protein turnover in mus- cle cells. The potential for autocrine and paracrine regulation of muscle growth, as well as the discovery of factors such as the APPENDIX differentiation inhibitor, emphasize the im- portance of efforts to isolate currently un- known peptide factors that significantly in- fluence the development of muscle tissue. In adclition to mitogenic growth factors, factors that inhibit the growth of cells have been reported (Hare] et al., 1985; Harring- ton ant! Gociman, 1980; Salmon et al., 19831. Although these factors have not been well characterized, it seems reasonable to as- sume that they modulate the growth-pro- moting effects of mitogenic serum factors such as the IGFs. In fact, both specific and nonspecific inhibitors of IGF action have been reported (Kuffer ancl lIerington, 1984; Salmon et al., 1983~. Although these inhib- itors have been detected in normal sera (Kuffer ant! Herington, 1984), their level and activity appear to be increased by cat- abolic conditions in both humans and ex- perimental animals (Salmon et al., 1983; Unterman and Phillips, 19851. Under the proper conditions, transforming growth fac- tor-,B (TGF-'B) has also been shown to inhibit proliferation of certain types of cultured cells (Roberts et al., 1985~. Because these inhibitory factors appear to have the poten- tial to attenuate the action of growth-pro- moting factors, it is important that more is learned about their mode of action and physiological significance in meat-pro(lucing animals. Radioimmunoassays (RIAs) cannot be used effectively to detect and characterize un- known or poorly characterized muscle growth factors. Consequently, bioassays capable of reliably detecting factors influencing muscle growth are necessary. These bioassays will augment existing RIAs by enabling us to detect ant] study currently unknown factors that may stimulate or inhibit muscle growth in meat-producing animals. The current lack of understan(ling of the mechanisms con- trolling muscle growth in meat animals is largely the result of (lifficulties encountered in devising a satisfactory bioassay system in which to study these processes. Experi- mental animals, isolated muscles, and mus
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BIOASSAYS cle cell culture have been the primary systems used to study the effects of specific peptides on the growth of muscle tissue. While experimental animals provide the most biologically complete system in which to study muscle growth, the complex inter- actions of their hormonal systems and large animal-to-animal variation often make it difficult to evaluate the role of any specific factor in muscle growth. Additionally, ex- periments with animals are expensive and labor intensive and often require several weeks or months to complete. In order to evaluate the effect of a specific factor on muscle growth, it is also necessary to meas- ure the muscle mass of control and experi- mental animals. At present, this is a labo- rious and inaccurate procedure. In vitro incubation of excised muscle tissue has also been used to study the effects of various peptides on muscle growth, pri- marily the influence of different substances on the rates of protein synthesis and deg- radation in skeletal muscle tissue (Fulks et al., 1975~. This technique provides a more controlled experimental environment and easier measurement of protein synthesis and degradation rates than does the whole ani- mal. However, excised muscles are gener- ally in a catabolic state relative to protein turnover (for example, protein degradation exceeds protein synthesis) (Clark and Mitch, 1983; Fulks et al., 1975~. Muscle cell culture has been used exten- sively to study the effects of specific DeDtides on both protein turnover and muscle cell proliferation. In culture, muscle precursor cells differentiate and proliferate to form myoblasts that fuse to form multinucleated myotubes. Myotubes synthesize contractile proteins, assemble them into myofibrils, and develop the ability to contract. How- ever, for these processes to occur, the culture media must contain blood serum or serum factors. Presumably, serum contains specific factors that are necessary for the differentiation and proliferation of muscle cells in culture. Consequently, muscle cell 177 culture has been used to study the effect of specific factors on proliferation, protein turnover, and differentiation in muscle cells. Although cell culture lends itself well to these kinds of studies, there is some concern about whether the findings are valid for muscle tissue in viva. Therefore, cell culture data must ultimately be confirmed in the animal. EFFECT OF PORCINE GROWTH HORMONE ON BIOACTIVITY AND IGF-I CONCENTRATION IN SWINE SERUM Although all the systems discussed in the preceding section may be useful as bioassays under the proper circumstances, my col- leagues and I have focused our efforts on developing and statistically standardizing a muscle cell culture bioassay that can be used to identify factors influencing muscle growth and to determine their mode of action in meat animals. This muscle cell culture bioassay and an IGF-I radioimmu- noassay have been used to measure the bioactivity and IGF-I concentration, re- spectively, in sera obtained from pigs before and after injection with porcine growth hormone (pGH). Although there have been conflicting re- ports about the effect of exogenous growth hormone (GH) on muscle growth in pitui- tary-intact swine, it now appears that long- term injection of highly purified pGH in- creases muscling, decreases fat, and im- proves feed efficiency in growing pigs (Chung et al., 1985; Machlin, 1972). However, very little is known about the mechanism through which pGH affects muscle deposition in pituitary-intact swine. Although it appears likely that the GH-induced increases in the circulating level of somatomedin-C may be responsible for increased muscle deposition, little information is available on the effect of artificially increased growth hormone lev- els on the concentration and bioactivity of somatomedins and other growth factors whose
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178 levels might be affected by this increase. Comparison of the muscle cell culture bioas- say response and the raclioimmunoassayable IGF-I concentration of sera obtained from pigs before and after pGH injection should help determine whether IGF is uniquely responsible for increases in muscle growth resulting from growth hormone treatment. Methods Standardized bioassays for measuring the elect of porcine serum on proliferation in cultured LO muscle cells were done accord- ing to procedures described in detail by Kotts et al. (1987a,b). Briefly, L6 cells were plated at 600/cm2 (25-cm2 flasks) in Dulbec- co's modified Eagle's medium (DMEM) containing 10 percent fetal calf serum. After 24 hours of attachment, the medium was removed and the cells were rinser! with 37°C DMEM without serum (SF media). Test media were applied ant] cells were incubated for 72 hours. The cells were removed for counting by trypsinization for 5 minutes at 37°C, and the reaction was stopper] by adding ice-col(1 DMEM con- taining 10 percent fetal calf serum. Cells from each flask were quantitatively trans- ferred to glass tubes on ice. The contents of each tube were diluted and counted in triplicate, and the counts were averaged. Triplicate flasks were assayed for each serum sample tested, ant! the results were ex- pressed as the mean number of cells/cm2 per flask + standard error. The intraassay coefficient of variation was 2.6 percent (Kotts et al., 1987b). Test media consisted of DMEM containing 3 percent (volume/volume) test sera. Porcine growth hormone was purchased from Dr. A. F. Parlow (Torrance, Calif.~. The pGH used for injection was lot no. 7024-C (specific activity = 1.5 U/mg) and that used for radioimmunoassay standard was lot no. APE 6400. i25I-pGH and rabbit anti-bovine GH were supplier! by Monsanto Company (St. Louis, Dog. Crossbred bar APPENDIX rows (19 to 36 kg) from separate litters were individually penner! and fee! ad libitum a corn- and soybean-basec] diet containing 21 percent protein. Five pigs were injected with 143 log of pGH/kg of body weight per clay for 3 clays. Catheters were inserted into both jugular veins, and after a 2-day recovery period, 12-ml blood samples were removed from the catheters at 6-hour intervals (6 a.m., noon, 6 p. m., and midnight) throughout the duration of the study. Injections of pGH were given at 2 p.m. on days 4 through 6. On days 1 through 3 and 7 through 9, all pigs received sham injections containing sterile saline. Injection and postinjection blooc! samples were collected on clays 4 through 9. The blood was allowed to clot, and serum was prepared for use in the muscle cell culture bioassays and radioim munoassays. Solutions of pGH for injection were pre- pared by dissolving the pGH in 44 mM NaHCO3, pH 11.5, and then immediately lowering the pH to 9.5 by addition of 1 N HC1. Solutions were prepared on the day of the first injections and filtered through a 0.22-,um filter. Protein content of the fil- tered solution was determined by the mi- crobiuret method. The basic electrophoresis system used for analytical sodium clodecy} sulfate (SDS) polyacrylamide slab gels was that of Laem- mli (1970) and consisted of a 3.5 percent acrylamide stacking gel ant! a 12 percent separating gel. Radioimmunoassays were done on the individual 6-hour serum samples obtained from each pig during the study. Radioim- munoassay kits from Micromedic Systems (Horsham, Pa.) were user! to quantify the levels of insulin and cortisol in the sera. The insulin kit was a homologous RIA for porcine insulin and used rabbit anti-porcine insulin antisera. The cortisol kit used rabbit anti-cortisol sera. A heterologous radioimmunoassay for porcine growth hormone was used to quan
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BIOASSAYS tify levels of pGH in the sera. This raclioim- munoassay used pGH (pituitary; lot AFP 6400) as a standard, i25I-pGH as a trace, and rabbit anti-bovine growth hormone anti- sera. The sensitivity of the assay at 95 percent binding was 5 ng/ml. Serial dilution of porcine serum at 100, 150, 200, and 250 ~1 yielded a curve that was parallel to the pGH standard curve. Recovery of standard in the presence of 200 ~1 of serum was 98.6 percent. The intraassay variability was 2.95 percent, and the interassay variability was 9.8 percent. All samples compared to each other in this work were assayed in the same experiment to avoic] interassay variation. Somatomedin-C levels in serum were quantified with a kit from the Nicholls Institute (San Juan Capistrano, Calif. ). Sera were treated in 1 M glycine-glycine HC1 buffer (pH 3.5) for 24 hours at 37°C prior to assay. All sera were measured against a human serum SM-C standard (1 U = 36 ng of purified SM-C). The trace was i25I-human SM-C; rabbit anti-human somatomedin-C antisera were used. The intraassay variabil- ity was 5.4 percent, and the interassay variability was 9.2 percent. When acidified swine serum was assayed in the presence of ~25I-human SM-C standard, 100 percent recovery was achieved. A titration of various dilutions (1:4 to 1:20) of swine sera resulted in curves parallel to those obtained with purified SM-C. To verify that the observed increases in mitogenic activity resulted from the oGH injections ant] were not random daily vari- ations in serum activity, the data obtained from the bioassay were subjected to analysis of variance. A randomized block design was used, with blocks representing pigs. To test for differences owing to pGH injection, the bioassay results from the preinjection days (1 through 3) were compared to those during (days 4 through 6) and after (days 7 through 9) injection by using the single degree of freedom contrasts on treatments. 179 Results and Discussion SDS-polyacrylamicle gel electrophoresis of the pGH preparation used in this study shower! a major band at 21.9 kilodaltons (Al) en cl a minor band at 20 kit, along with several minor hands between 15 anal 9 kd. The molecular weights of the 21.9- and 20- k] bands correspond to those reported for human growth hormone (Chambach et al., 1973~. The peptides banding between 9 and 15 k(l may be proteolytic fragments of pGH, or they may be impurities in the prepara- tion. Whatever their origin, any single one of these peptides represents an extremely minor contaminant in the pGH preparation. To determine whether the pGH prepa- ration contained contaminants that affected muscle cell proliferation, it was added at various concentrations to media containing 2.5 percent (volume/volume) control swine serum (CSS). Radioimmunoassay of the CSS showed that it contained 5.56 ng of pGH/ m} and 2.18 U of SM-C/mI. Consequently, the contribution of the CSS to the final pGH or SM-C level in the bioassay was 6 x 10-12 M pGH and 2.58 x 10-1° M SM- C (based on a molecular weight of 7.6 kd and 36.1 ng of human SM-C/U and a mo- lecular weight of 22 Ed for pGH). The proliferation rate of cultured muscle cells was not significantly affected by pGH con- centrations below 10-8 M, but 10-8 M pGH or higher resulted in a slight, though sig- nificant, increase in cell numbers (10 to 12 percent above control levels). The inability of pGH to stimulate proliferation of cultured muscle cells is in agreement with results obtained by others using primary myogenic cultures or L6 myogenic cells (Ewton and Florini, 1980; Gospodarowicz et al., 1976~. The slight stimulation of proliferation ob- served at higher pGH concentrations (~10-8 M) is consistent with the stimulation of alpha-aminoisobutyric acid uptake in 8-day- old cultures of L6 myotubes exposed to 10 - 7
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180 M bovine GH (Ewton and Florini, 1980~. It is possible that impurities in the GH preparation or biologically active fragments of the GH molecule (Liberti and Miller, 1978) are responsible for these increases in mitogenic activity observed at supraphy- siological concentrations of GH. In contrast to the lack of response ob- served when pGH was aciclec3 directly to muscle cells, sera from four out of five pigs injected with pGH exhibiter] increased mi- togenic activity. Analysis of variance on the bioassay data from all five pigs showed that the treatment elects were highly significant (P < 0.005~. The single degree of freedom contrasts on treatment revealer! that the mitogenic activity of sera obtained during and after the pGH injections was signifi- cantly higher (P < 0.005) than preinjection levels. Aciclitionally, all pigs receiving pGH showed increases in SM-C levels in their sera during and after the injections. The pGH concentration in the 24-hour pooled serum samples from the pigs on pGH injection days (clays 4 through 6) was approximately 100 ng/ml, and these pools were diluted 29-fold for use in the prolif- eration bioassay. Thus, the maximum con- centration of pGH in the bioassay media was 10-~° M. Since 10-~° M pGH had no elect on proliferation when added directly to the muscle cell cultures, the increases in bioassayable mitogenic activity of serum pools obtained cluring ant] after pGH injec- tion were not a direct result of the increased level of pGH in the culture media. Serum pGH levels were increased ap- proximately 30-folcl by 4 hours after each pGH injection and declined to preinjection levels by approximately 16 hours after each injection. Increases in serum SM-C levels were observed 6 to 12 hours after the increase in serum pGH concentration (10 to 16 hours after each pGH injection). The magnitude of the SM-C response was dif- ferent for each pig, even though all pigs received the same dose of pGH and attained similar blood levels of pGH 4 hours after APPENDIX injection. SM-C increases ranged from 1.7 to 4 times the preinjection levels. In all the pigs, the second and third! injections re- sulted in higher concentrations of serum SM-C than the first injection. In two cases, SM-C concentrations appeared to increase in a stepwise manner with each successive injection of pGH. A similar stepwise in- crease in SM-C production upon successive injections of human growth hormone into hypopituitary patients was reported by Copeland et al. (1980~. Serum SM-C levels remained high for 2 to 6 days after the last pGH injection. Insulin en c! cortiso} levels in the sera dicI not change during the treatment period ant! ranged from 3.8 to 10.6 ~U/ml and 2.0 to 6.9 ~g/~l, respectively. It is well established that GH stimulates the production of somatomedins (IGFs) by the liver and possibly by other tissues as well. Administration of IGF-I/SM-C to hy- pophysectomized rats has been reported to restore growth to a level equivalent to that seen with GH replacement (SchoenIe et al., 1982~. Additionally, IGF-I/SM-C and IGF- II/MSA stimulate the proliferation of my- ogenic cells in culture (Ballard et al., 1986; Ewton en cl Florini, 1981; Florini et al., 1984; Hill et al., 1986a). Consequently, it appears likely that the increased levels of IGF-I/SM-C observed in sera obtained from pigs cluring and after pGH injection play a role in the increased mitogenic activity of these sera. Nonetheless, there were several instances when changes in serum IGF-I/ SM-C levels did not appear to be directly related to changes in serum mitogenic ac- tivity in the bioassay. For example, sera from pig 90 showed a significant increase in SM-C concentration cluring and after pGH injection (2.5 U/ml preinjection to 6.5 U./ ml postinjection); however, no correspond- ing increase in serum mitogenic activity was detectable. In contrast, sera from pig 85 exhibited a similar change in serum SM-C concentration during and after pGH injec- tion (2 U/m! preinjection to 7 U/m} postin
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BIOASSAYS jection), and this corresponded to a signif- icant increase in mitogenic activity. In acI- dition, sera from pig 87 exhibiter! a relatively large increase in SM-C concentration (3.5 U/m! preinjection to 10 to 13 U/ml postin- jection) but shower] only a modest increase in mitogenic activity. Conversely, sera from pig 7, which exhibited relatively little in- crease in SM-C concentration (2 U/ml prein- jection to 4 to 5.5 U/ml postinjection), showed a relatively large increase in mito- genic activity (24 percent) over the injection - period. These results suggest that factors in acI- dition to radioimmunoassayable IGF-I/SM- C may contribute to the alterations in mi- togenic activity observed in sera during and after pGH injection. There are several fac- tors that could! be involved in the mitogenic response, either by directly affecting muscle cell proliferation or by modulating the bioac- tivity of IGF-I. For example, IGF-II has been reported to increase fourfold in the sera of GH-deficient humans after GH administration (Schalch et al., 1982~. Ad- clitionally, inhibitors of IGF-stimulated syn- thesis of DNA anchor sulfate incorporation in costar cartilage have been reported in sera from starved, diabetic, or hypophysec- tomized rats (Kuffer and Herington, 1984; Salmon et al., 1983; Unterman and Phillips, 1985), and a specific inhibitor of IGF has been isolated ant! partially purified from normal sera (Kuffer and Herington, 1984~. Somatomeclin-binding proteins ranging in molecular weight from 40 to 70 kit have also been reported to bind and inactivate IGF (Hossenlopp et al., 1986; Martin and Baxter, 1985; Romanus et al., 1986~. In addition, a protein that inhibits differentiation of my- ogenic cells has been identified in fetal calf serum and in merlin obtained from BRL cells in culture (Evinger-Hodges et al., 1982; Florini et al., 1984~. It is possible that these factors or other, as yet unidentified, factors are affecting the mitogenic activity of sera in the muscle cell culture bioassay used in this study. 181 Results of this study demonstrate the importance of developing bioassays for mus- cle growth. Used in conjunction with ra- dioimmunoassays, bioassays can help elu- cidate the mode of action of known growth factors such as somatotropin. They also provide a valuable tool for use in identifying unknown growth factors that affect muscle growth in meat animals. Identification of these factors ant] clarification of their mode of action is crucial to an eventual under- stancling of the biological control of muscle growth. REFERENCES Adams, S. O., M. Kapadia, B. Mills, and W. H. Daughaday. 1984. Release of insulin-like growth factors and binding protein activity into serum-free medium of cultured human fibroblasts. Endocrinol- ogy 115:520. Albertsson-Wikland, K., and 0. Isaksson. 1976. De- velopment of responsiveness of young normal rats to growth hormone. Metabolism 25:747. Allen, R., K. C. Masak, P. K. McAllister, and R. A. Merkel. 1983. Effects of growth hormone, testos- terone and serum concentration on actin synthesis in cultured satellite cells. J. Anim. Sci. 56:833. Allen, R. E., M. V. Dodson, and L. S. Luiten. 1984. Regulation of skeletal muscle satellite cell prolifer- ation by bovine pituitary fibroblast growth factor. Exp. Cell Res. 152:154. Ballard, F. J., L. C. Read, G. L. Francis, C. J. Bagley, and J. C. Wallace. 1986. Binding properties and biological potencies of insulin-like growth factors in L6 myoblasts. Biochem. J. 233:223. Breuer, C. B. 1969. Stimulation of DNA synthesis in cartilage of hypophysectomized rats by native and modified placental lactogen and anabolic hormones. Endocrinology 85:989. Chambach, A., R. A. Yadley, M. Ben-David, and D. Rodbard. 1973. Characterization of human growth hormone by electrophoresis and isoelectric focusing in polyacrylamide gel. Endocrinology 93:848. Chung, C. S., T. D. Etherton, and J. P. Wiggins. 1985. Stimulation of swine growth by porcine growth hormone. J. Anim. Sci. 60:118. Clark, A. S., and W. E. Mitch. 1983. Comparison of protein synthesis and degradation in incubated and perfused muscle. Biochem. J. 212:649. Copeland, K. C., L. E. Underwood, and J. J. Van Wyk. 1980. Induction of immunoreactive somato- medin-C in human serum by growth hormone: Dose response relationships and effects on chromato- graphic profiles. J. Clin. Endocrinol. Metab. 50:690.
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182 Davis, S. L., U. S. Garrigus, and F. C. Hinds. 1969. Metabolic effects of growth hormone and diethyl- stilbestrol in lambs. II. Effects of daily ovine growth hormone injections in plasma metabolites and nitro- gen-retention in fed lambs. J. Anim. Sci. 30:236. Dollenmeier, P., D. C. Turner, and H. M. Eppen- berger. 1981. Proliferation of chick skeletal muscle cells cultured in a chemically defined medium. Exp. Cell Res. 135:47. Evinger-Hodges, M. J., D. Ewton, Z. S. C. Seifert, and J. R. Florini. 1982. Inhibition of myoblast differentiation in vitro by a protein isolated from liver cell medium. J. Cell Biol. 93:395. Ewton, D. Z., and J. R. Florini. 1980. Relative effects of the somatomedins, MSA and growth hormone on myoblasts and myotubes in culture. Endocrinology 106:577. Ewton, D. Z., and J. R. Florini. 1981. Effects of the somatomedins and insulin on myoblast differentia- tion in vitro. Dev. Biol. 86:31. Florini, J. R., and D. Z. Ewton. 1981. Insulin acts as a somatomedin analog in stimulating myoblast growth in serum-free medium. In Vitro 17:763. Florini, J. R., and S. B. Roberts. 1979. A serum-free medium for the growth of muscle cells in culture. In Vitro 15:983. Florini, J. R., D. Z. Ewton, M. J. Evinger-Hodges, S. L. Fallen, R. L. Lau, J. F. Ragan, and B. M. Vertel. 1984. Stimulation and inhibition of myoblast differentiation by hormones. In Vitro 20:942. Fulks, R. M., J. B. Li, and A. L. Goldberg. 1975. Effects of insulin, glucose and amino acids on protein turnover in rat diaphragm. J. Biol. Chem. 250:290. Gospodarowicz, D., J. Weseman, J. S. Moran, and J. Lindstrom. 1976. Effect of fibroblast growth factor on the division and fusion of bovine myoblasts. J. Cell Biol. 70:395. Harel, L., C. Blat, and G. Chatelain. 1985. Regulation of cell proliferation inhibitory and stimulatory factors diffused by 3T3 cultured cells. J. Cell. Physiol. 123:139. lIarrington, W. N., and G. C. Godman. 1980. A selective inhibitor of cell proliferation from normal serum. Proc. Natl. Acad. Sci. USA 77:423. Hill, D. J., C. J. Crace, S. P. Nissley, D. Morrell, A. T. Holder, and R. D. G. Milner. 1986a. Fetal rat myoblasts release both rat somatomedin-C (SM-C)/ insulin-like growth factor I (IGF I) and multiplica- tion-stimulating activity in vitro: Partial characteri- zation and biological activity of myoblast-derived SM-C/IGF I. Endocrinology 117:2061. Hill, D. J., C. J. Crace, A. J. Strain, and R. D. G. Milner. 1986b. Regulation of amino acid uptake and deoxyribonucleic acid synthesis in isolated human fetal fibroblasts and myoblasts: Effect of human placental lactogen, somatomedin-C, multiplication stimulating activity, and insulin. J. Clin. Endocrinol. Metab. 62:753. APPENDIX Hossenlopp, P., D. Seurin, B. Segoria-Quinson, S. Hardouin, and M. Binoux. 1986. Analysis of serum insulin-like growth factor binding proteins using Western blotting: Use of the method for titration of the binding proteins and competitive binding stud- ies. Anal. Biochem. 154:138. Ii, I., I. Kimura, T. Hasegawa, and E. Ozawa. 1981. Transferrin is an essential component of chick embryo extract for avian myogenic cell growth in vitro. Proc. Jpn. Acad. 57:211. Janeczko, R. A., and J. D. Etlinger. 1984. Inhibition of intracellular proteolysis in muscle cultures by multiplication-stimulating activity. Comparison of effects of multiplication-stimulating activity and in- sulin on proteolysis, protein synthesis, amino acid uptake, and sugar transport. J. Biol. Chem. 259:6292. Jefferson, L. S., J. B. Li, and S. R. Rannels. 1977. Regulation by insulin of amino acid release and protein turnover in perfused rat hemicorpus. Proc. Natl. Acad. Sci. USA 69:816. Karkami, E., D. Spector, and R. C. Strohman. 1985. Myogenic growth factor present in skeletal muscle is purified by heparin-affinity chromatography. Proc. Natl. Acad. Sci. USA 82:8044. King, G. L., C. R. Kahn, M. M. Rechler, and S. P. Nissley. 1980. Direct demonstration of separate receptors for growth and metabolic activities of insulin and multiplication-stimulating activity (an insulin-like growth factor) using antibodies to the insulin receptor. J. Clin. Invest. 66:130. Kotts, C. E., M. E. White, C. E. Allen, and W. R. Dayton. 1987a. Stimulation of in vitro muscle cell proliferation by sera from swine injected with porcine growth hormone. J. Anim. Sci. 64:623. Kotts, C. E., M. E. White, F. Martin, C. E. Allen, and W. R. Dayton. 1987b. A statistically standard- ized bioassay for measuring the proliferation rate of myogenic cells in culture. I. Anim. Sci. 64:615. Kuffer, A. D., and A. C. Herington. 1984. Partial purification of a specific inhibitor of the insulin-like growth factors by reversed-phase high-performance liquid chromatography. J. Chromatogr. 336:87. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680. Liberti, J. P., and M. S. Miller. 1978. Somatomedin- like effects of biologically active bovine growth hormone fragments. Endocrinology 103:680. Linkhart, T. A., C. H. Clegg, and S. D. Hauschka. 1981. Myogenic differentiation in permanent clonal mouse myoblast cell lines: Regulation by macro- molecular growth factors in the culture medium. Dev. Biol. 86:19. Machlin, L. J. 1972. Effect of porcine growth hormone on growth and carcass composition of the pig. J. Anim. Sci. 35:794. Marquardt, H., G. J. Todaro, L. E. Henderson, and S. Oroszlan. 1981. Purification and primary structure
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BIOASSAYS of a polypeptide with multiplication-stimulating ac- tivity from rat liver cell cultures. J. Biol. Chem. 256:6859. Martin, J. L., and R. C. Baxter. 1985. Antibody against acid-stable insulin-like growth factor binding protein detects 150,000 mol wt growth hormone-dependent complex in human plasma. J. Clin. Endocrinol. Metab. 61:799. Massague, J., and M. P. Czech. 1982. The subunit structures of two distinct receptors for insulin-like growth factors I and II and their relationship to the insulin receptor. J. Biol. Chem. 257:5038. Matsuda, R., D. Spector, and R. C. Strohman. 1984. There is selective accumulation of a growth factor in chicken skeletal muscle. I. Transferrin accumu- lation in adult anterior latissimus dorsi. Dev. Biol. 103:267. Monier, S., and Y. Le Marchand-Brustel. 1984. Effects of insulin and IGF-I on RNA synthesis in isolated soleus muscle. Mol. Cell. Endocrinol. 37:109. Moseley, W. M., L. F. Krabill, and R. F. Olsen. 1982. Effect of bovine growth hormone administered in various patterns on nitrogen metabolism in the Holstein steer. J. Anim. Sci. 55:1062. Ozawa, E., and K. Kohama. 1978. Partial purification of a factor promoting chicken myoblast multiplication in vitro. Proc. Jpn. Acad. 49:852. Pain, V. M., and P. J. Garlick. 1974. Effect of strep- tozotocin diabetes and insulin treatment on the rate of protein synthesis in tissues of the rat in vivo. J. Biol. Chem. 249:4510. Peel, C. J., D. E. Bauman, R. C. Gorewit, and C. J. Sniffen. 1981. Effect of exogenous growth hormone on lactational performance in high yielding dairy cows. J. Nutr. 111:1662. Roberts, A. M., M. A. Anzano, L. M. Wakefield, N. S. Roche, D. F. Stern, and M. B. Sporn. 1985. Type beta transforming growth factor: A bifunctional 183 regulator of cellular growth. Proc. Natl. Acad. Sci. USA 82:119. Romanus, J. A., J. E. Terrell, Y. W.-H. Yang, S. P. Nissley, and M. M. Rechler. 1986. Insulin-like growth factor carrier proteins in neonatal and adult rat serum are immunologically different: Demon- stration using a new radioimmunoassay for the carrier protein from BRL-3A rat liver cells. Endocrinology 118:1743. Salmon, W. D., L. A. Holladay, and V. J. Burkhalter. 1983. Partial characterization of somatomedin inhib- itors in starved rat serum. Endocrinology 112:360. Schalch, D. S., S. E. Tollefsen, G. J. Klingensmith, R. W. Gotlin, and M. J. Diehl. 1982. Effects of growth hormone administration on serum somato- medins, somatomedin carrier proteins and growth rates in children with growth hormone deficiency. J. Clin. Endocrinol. Metab. 55:49. Schoenle, E., J. Zapf, R. E. Humbel, and E. R. Froesch. 1982. Insulin-like growth factor I stimulates growth in hypophysectomized rats. Nature 296:252. Shen, V., L. Rifas, G. Kohler, and W. Peck. 1985. Fetal rat chondrocytes sequentially elaborate sepa- rate growth- and differentiation-promoting peptides during their development in vitro. Endocrinology 116:920. Shimo-Oka, T., Y. Hagiwara, and E. Ozawa. 1986. Class specificity of transferrin as a muscle trophic factor. J. Cell. Physiol. 126:341. Unterman, T. G., and L. S. Phillips. 1985. Glucocor- ticoid effects on somatomedins and somatomedin inhibitors. J. Clin. Endocrinol. Metab. 61:618. Wagner, J. F., and E. L. Veenhuien. 1978. Growth performance, carcass deposition and plasma hor- mone levels in wether lambs when treated with growth hormone and thyroprotein. J. Anim. Sci. 46(Suppl. 1):397.
Representative terms from entire chapter: