Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter.
Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.
OCR for page 135
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;
OCR for page 136
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.
OCR for page 137
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~.
OCR for page 138
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
OCR for page 139
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
OCR for page 140
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.
, ,
OCR for page 141
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.
OCR for page 142
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
OCR for page 143
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!
OCR for page 144
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
OCR for page 145
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
OCR for page 173
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
OCR for page 174
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
OCR for page 175
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
OCR for page 176
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
OCR for page 177
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
OCR for page 178
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
OCR for page 179
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
OCR for page 180
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
OCR for page 181
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.
OCR for page 182
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
OCR for page 183
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:
muscle growth
