247
NUTRITIONAL REGULATION OF MACROM0I;ECULAR SYNTHESIS I N MUSCLE*
IBKNER G . BERGEIi
Michigan S t a t e University
INTRODUCTION
Detailed reviews on t h e o v e r a l l mechanisms of p r o t e i n synthesis
( t r a n s l a t i o n ) have r e c e n t l y been completed by Lucas-Lenard and Lipmann
( 1971), Haselkorn and Rothman-Denes ( 1973) Ochoa and Mazumder (1974 )
Lucas-Lenard and Beres (1974) and Tate and Caskey (1974). Young (1970,
1974) and Bergen (1974) have published reviews on t h e r e g u l a t i o n of
p r o t e i n synthesis and growth i n mammalian muscle. The present review
w i l l place emphasis on some of t h e m a t e r i a l covered above and w i l l a l s o
focus on some of the more recent research (and new problems) i n p r o t e i n
synthesis regulation, e s p e c i a l l y as it a p p l i e s t o muscle.
,
,
The following t o p i c s w i l l be covered:
(1) Regulation of p m t e i n synthesis i n muscle (eukaryotic) c e l l s .
(2)
I n i t i a t i o n , s p e c i f i c i t y of i n i t i a t i o n f a c t o r s and mRNA
binding, elongation and coordinate synthesis of t h e various
muscle p r o t e i n s .
( 3 ) Localization o f ribosomes i n
t h e m y D f i b e r and t h e i r p o t e n t i a l
r o l e i n t h e s p e c i f i c i t y f o r m y o f i b r i l l a r and soluble muscle
p r o t e i n synthesis.
(4)
Muscle p r o t e i n turnover.
(5)
The r o l e of developmental and o t h e r hormones on muscle
protein synthesis.
(6)
Overall developmental aspects, c e l l u l a r i t y , nucleic acid
metabolism and e f f i c i e n c y of p r o t e i n synthesis during muscle
growth.
( 7 ) Problems i n studying t h e r o l e of n u t r i t i o n and hormones i n
muscle p r o t e i n synthesis mechanisms.
*
Presented a t t h e S t h Annual Reciprocal Meat Conference o f t h e American
Meat Science Association, 1975.
248
Regulation of protein synthesis i n muscle (eukaryotic) c e l l s
Extensive work of t h e mechanism of protein synthesis has been
conducted w i t h b a c t e r i a l (prokaryotic ) systems. Protein synthesis can
be divided i n t o two areas: t r a n s c r i p t i o n , i . e . , the DNA d i r e c t e d
generation of genetic messages (mRNA) and t h e synthesis of the machinery
f o r p r o t e i n synthesis (rRNA and tRNA); and t r a n s l a t i o n , i . e . , the
decoding of t h e message i n t o p r o t e i n s .
Translation can be divided i n t o 3 separate steps: I n i t i a t i o n ,
elongation and termination. I n i t i a t i o n involves t h e mechanisms of mRNA
binding t o t h e small ribosomal subunit and t h e recruitment of the
i n i t i a t o r tRNA (Met-t-RNAf) and the f i n a l binding of t h e l a r g e r i b o somes subunit. This process i s then followed by elongation, the stepwise addition of a s i n g l e amino acid ( v i a AA-tRNA) i n t o the s t a r t e d
peptide chain as t h e message i s decoded. Finally, upon completion ( o r
upon reaching a termination codon), t h e macromolecule i s r e l e a s e d .
This process i s c a l l e d termination.
Haselkorn and Rothman-Denes (1973) concluded t h a t t r a n s l a t i o n a l
mechanisms of p r o t e i n synthesis a r e r e l a t i v e l y s i m i l a r between prokaryotic
and eukaryotic c e l l s . Based on t h e work of Heywood, Rich and coworkers
(Heywood e t a l . , 1967; Heywood e t al., 1968) it has been concluded t h a t
t h e o v e r a l l mechanisms of s k e l e t a l muscle protein synthesis a r e s i m i l a r
t o t h e process extensively described f o r b a c t e r i a l or l i v e r systems.
A number of f a c t o r s t h a t regulate o r might be r a t e l i m i t i n g i n
muscle protein synthesis as enumerated by Bergen (1974) and Young (1974)
a r e l i s t e d below:
(1) Ribosome a v a i l a b i l i t y and competence
(2)
h u n t of messenger KNA
(3)
Substrate supply (AA)
( 4 ) Amino acyl-tRNA
( 5 ) Soluble f a c t o r s and other f a c t o r s and enzymes
( 6 ) Hormones and hormonal l i k e f a c t o r s
A major long term f a c t o r i n t h e regulation of protein synthesis i s
t h e a v a i l a b i l i t y and competence of ribosomes (rRNA) (Wannemacher, 1972;
Henshaw e t a l . , 1971). The amount of t i s s u e rRNA i s d i r e c t l y r e l a t e d
t o t h e s u b s t r a t e supply and t h e r e i s a high correlation between t i s s u e
RNA l e v e l and r a t e of p r o t e i n synthesis (Wannemacher, 1972; Allison et
1963; Wannemacher e t al., 1971). When t h e d i e t a r y amino acid
supply i s r e s t r i c t e d t o a growing r a t , muscle RNA declines and protein
synthesis declines (Howarth, 1972). Henshaw e t a l . , (1971) studied
protein synthesis in l i v e r and muscle of fed o r fasted r a t s . These
workers concluded t h a t a decrease i n protein synthesis t o f a s t i n g was
e.,
249
due t o a decrease i n c e l l u l a r RNA content a s w e l l as polysomal a c t i v i t y
and t h e proportion o f ribosomes i n polysomes. Von der Decken and Omstedt
(1970, 1972) and Omstedt and Von d e r Decken (1972) studied t h e e f f e c t of
p r o t e i n s t a r v a t i o n and n u t r i t i v e q u a l i t y (Mpu) of d i e t a r y proteins on
i n v i t r o i n r a t s k e l e t a l muscle. It w a s demonstrated
prDtein synthesis -t h a t when an inadequate p r o t e i n d i e t ( e i t h e r l e v e l o r n u t r i t i v e q u a l i t y )
was fed t o r z t s , t h e r e was a decrease i n ribosomal RNA i n t h e muscle
t i s s u e as w e l l a s i n t h e a c t i v i t y of t h e ribosomes.
Some other n u t r i t i o n a l f a c t o r s have been r e l a t e d t o p r o t e i n synthesis
i n muscle c e l l s . Protein synthesis i n perfused rat h e a r t muscle was
enhanced by oxidizable non carbohydrate s u b s t r a t e s (Rannels e t a l . ,
1974). A vitamin E deficier'cy i n r a b b i t s caused s h i f t s i n t h e synthesis
of s p e c i f i c muscle proteins ( S h a r d and Srivastava, 1974).
The amount of a v a i l a b l e mRNA can a l s o c o n t r o l o v e r a l l p r o t e i n
synthesis; t h i s has been e s p e c i a l l y w e l l demonstrated i n b a c t e r i a l
systems which have mRNA w i t h s h o r t h a l f - l i f e (Haselkorn and RothmanDenes, 1973). When p r o t e i n synthesis commences t h i s can u s u a l l y be
demonstrated by an aggregation of ribosomes on mRNA which r e s u l t s i n
polysome formation (Munro, 1970). Unfortunately the aggregation of
ribosomes i n t o polysames may be i n t e r p r e t e d t o be due t o synthesis of
new mRNA species or a n enhanced recruitment of already e x i s t i n g mRNA
and possibly o t h e r f a c t o r s . I n extensive work with t h e chick oviduct
system and t h e e f f e c t of various hormones ( e .g estrogen, progesterone
and t e s t o s t e r o n e ) on the increase i n t h e synthesis r a t e of s p e c i f i c
p r o t e i n s , it has been shown that t h e r e may be a n increase i n IuREA
s y n t h e s i s . There a l s o may be an increase i n DNA dependent RNA polymerase a c t i v i t y a s w e l l as a more e f f i c i e n t recruitment of mRNA (increased
rate of i n i t i a t i o n and elongation) (Mears and O'Malley, 1971; Palmiter,
1973; 1973; Palmiter and H a i n e s , 1973).
.
Myosin mRNA has been i s o l a t e d from chick embryo muscle and p a r t i a l l y
characterized (Morris e t g . , 1973). T h i s group of workers previously
(Heywood and Rich, 19687 showed t h a t t h e proportion of t h e polysome
synthesizing myosin increased (possibly because of increased mRNA
a v a i l a b i l i t y ) between t h e 9th and 17th day of development of chick
embryo muscle. This increase i n myosin synthesizing polysomes c l o s e l y
resembles t h e p a t t e r n of t h e appearance of t h i c k filaments and myosin
a c c r e t i o n i n the developing c e l l ( H e r m n n e t a1
1970). An increase
i n DNA dependent RNA polymerase has been obtained i n cardiac and s k e l e t a l
muscle undergoing hypertrophy and increased myosin synthesis (Scheiber
&
1969). Further t h e enhaacing e f f e c t o f growth hormone on
p r o t e i n synthesis i n muscle was i n p a r t ascribed t o changes i n RNA
polymerase a c t i v i t y ( F l o r i n i and Breuer, 1966). The above r e s u l t s
i n d i c a t e t h a t mRNA a v a i l a b i l i t y plays a major regulatory r o l e i n muscle
p r o t e i n synthesis.
.,
e.,
Young (1974) has reviewed t h e l i t e r a t u r e on t h e r o l e of aminoacylt R N A on p r o t e i n synthesis i n muscle. Upon d i e t a r y p r o t e i n depletion,
muscle aminoacyl-tRNA synthetase a c t i v i t y did not decrease (Young,
1974); hmever, t h e a c t i v i t y of t h i s enzyme decreased i n t h e l i v e r
(Stephen, 1968). Others have speculated on t h e r o l e cJf t h e 3 ' - t r i nucleotide and sequence ( - p c p c p ~ )on amino acid accepting capacity of
tRNA (Deutscher, 1973). Although d e f i n i t i v e r e s u l t s have n o t been
published f o r muscle c e l l s , t h e r e i s t h e d i s t i n c t p o s s i b i l i t y t h a t t h e
r a t e of p r o t e i n synthesis i n muscle c e l l s may be modulated by t h e
a v a i l a b i l i t y o f tRNA which can accept amino a c i d s (Young, 1974). The
role of s o l u b l e ( i n i t i a t i o n , elongation, termination and i n t e r f e r e n c e )
f a c t o r s and developmental and o t h e r hormones w i l l be discussed i n t h e
t o p i c sections below.
I n i t i a t i o n , s p e c i f i c i t y of i n i t i a t i o n f a c t o r s and mRNA binding, elongation
and coordinate synthesis of various muscle p r o t e i n s .
Three s e t s of p r o t e i n f a c t o r s a r e involved i n t h e process of
p r o t e i n synthesis and i n t h e proper decoding of t h e genetic message.
When i s o l a t e d ribosomes a r e washed with NH4Cl o r KC1, t h r e e p r o t e i n
f a c t o r s are removed (Lucas-Lenard and Lipman, 1971). The absence o f
these f a c t o r s , t r a n s l a t i o n of n a t u r a l mRNA i n t h e presence of ribosomes,
GTP, tRNA, amino a c i d s , ATP e t c
i s a l s o blocked.
.,
Since t h e i n i t i a t i o n process must involve a discriminatory mechanism
t o c o n t r o l which p r o t e i n s a r e t o be synthesized, (e.g. t r a n s l a t i o n a l
c o n t r o l ) extensive work has been conducted on t h e r o l e of i n i t i a t i o n
f a c t o r s on t h e o v e r a l l r a t e and products i n c e l l f r e e , n a t u r a l messenger,
p r o t e i n synthesis systems. I n t e r e s t i n elongation f a c t o r s i n muscle
work has centered on t h e r o l e of s u b s t r a t e supply on t h e elongation
cycle and p r o t e i n synthesis r a t e .
Although it had been g e n e r a l l y accepted t h a t t h e r e g u l a t i o n of
p r o t e i n synthesis occurs p r i m a r i l y a t t h e l e v e l of t r a n s c r i p t i o n ,
recent evidence i n prokaryotic ( a s w e l l as eukaryotic) systems has
suggested t h a t p r o t e i n synthesis may a l s o be controlled during t h e
t r a n s l a t i o n s t a g e - e s p e c i a l l y i n i t i a t i o n . There a r e t h r e e i n i t i a t i o n
f a c t o r s , e.g., IF1, IF2, IF3 (Lucas-Lenard and Lipman, 1971). During
i n i t i a t i o n , IF3 promotes t h e binding of n a t u r a l mRNA t o t h e small r i b o soml subunit (30s o r 40s) (Wefssbach and Brot, 1974; Vermeer e t a 1
1973). This binding of mRNA t o t h e small subunit i s stimulated about
two f o l d by IF2. This a c t i o n forms a s t a b l e ribosomal IF3-30s ( o r 40s)
mRNA complex. IF3 also appears t o promote a conformational change i n
t h e small subunit t o prevent l a r g e subunit ( 5 0 o r 60s) from binding
(Weissbach and Brot, 1974). The r o l e of t h e ribosome and IF3 i n t h e
binding of t h e i n i t i a t o r codon (AUG) i s p r e s e n t l y a very a c t i v e area
of research. IF1 and IF2 then f a c i l i t a t e t h e binding of t h e f-Met-tRNAf
( o r Met-tRNAf i n eukaryotes) i n t h e presence of GTP t o t h e IF3 small
subunit mRNA complex. IF2 i s h i g h l y s p e c i f i c f o r binding o f t h e
i n i t i a t o r Met-tRNAf and w i l l d i s c r i m i n a t e a g a i n s t t h e Met-tRNAm used
-
251
fm- i n t e r v a l methionine decoding. A s t h e Met-tRNAf i s bound t o t h e
complex, IF3 i s released, then f i n a l l y t h e GTP i s hydrolyzed, t h e l a r g e
subunit j o i n s t h e small subunit and I F 1 and IF2 a r e released (Weissbach
and Brot, 1974).
I n prokaryotic systems, IF3 appears t o recognize d i f f e r e n t c i s t r o n
i n i t i a t i o n s i t e s on p o l y c i s t r o n i c messages (Revel e t a1
1970; Berrassi
e t g . , 1971). In addition, a p r o t e i n , c a l l e d t h e i n t e r f e r e n c e f a c t o r
(if a c t o r ) has been i s o l a t e d (Groner e t a1 1972). The i f a c t o r
depresses t h e r e a c t i v i t y of IF3 w i t h c e r t a i n n a t u r a l W A .
.,
--.,
Heterologous and homologous t r a n s l a t i o n a l systems (embryonic
muscle, r e t i c u l o c y t e s ) have been used t o study t h e s p e c i f i c i t y of mRNA
binding. Tihen r e l a t i v e l y small amounts o f myosin mRNA o r myoglobin
mRNA were i n i t i a t e d w i t h heterologous ribosomes, ribosomal ( i n i t i a t i o n )
f a c t o r s were required from t h e same c e l l type a s t h e messenger t o i n s u r e
t al . , 1973). It was
decoding (Thompson and Heywood, 1974; Thompson esubsequently shown t h a t IF3 was responsible i n some manner i n mRNA
recognition (Heywood e t a l . , 1974). More r e c e n t l y a low molecular
w e i g h t RNA has been i s o l a t e d from i n i t i a t i o n f a c t o r (ribosomal s a l t
washes) which appears t o be involved i n t r a n s l a t i o n a l regulation of
p r o t e i n synthesis (Heywood e t a l . , 1974; Bogdanousky e t a l . , 1973).
This RNA c a l l e d t r a n s l a t i o n a l c o n t r o l RNA (tcRNA), i s not e f f e c t i v e
i n blocking mRNA binding t o ribosomes ( i n i t i a t i o n ) i n homologous systems,
b u t muscle tcRNA causes an abortive i n i t i a t i o n of globin synthesis i n
r e t i c u l o c y t e lysate systems (Kennedy e t a l . , 1974).
--
--
The r o l e of elongation f a c t o r s i n muscle p r o t e i n synthesis has been
primarily studied during p r o t e i n depletion. Young and coworkers (1974)
have concluded t h a t elongation f a c t o r s (EF) l i m i t p r o t e i n synthesis i n
muscle c e l l f r e e -i n v i t r o systems from p r o t e i n depleted r a t s . A r a t e
l i m i t i n g r o l e in vivo of EF i n muscle p r o t e i n synthesis has not been
e s t a b l i s h e d (Young, 1974) e s p e c i a l l y s i n c e a l l c e l l f r e e muscle p r o t e i n
synthesis systems do not e x h i b i t de novo r e i n i t i a t i o n , and t h e r o l e of
i n i t i a t i o n cannot be ruled o u t . Further, t h e high i o n i c s t r e n g t h
b u f f e r s necessary f o r ribosome removal i n muscle removes some of t h e
i n i t i a t i o n f a c t o r s ( I F ) from t h e polysome, and t h u s may o b l i t e r a t e
p o t e n t i a l d i f f e r e n c e s i n i n i t i a t i o n rates between muscle c e l l s from
normal and p r o t e i n depleted animals. It would seem most l o g i c a l t h a t
i n i t i a t i o n and maybe I F should be r a t e l i m i t i n g during periods of
protein depletion.
Much emphasis has been placed on research on t h e r e l a t i v e synthesis
and degradation r a t e o f l i g h t and heavy chains of myosin. I n embryonic
s k e l e t a l muscle homogenate systems, it w a s demonstrated t h a t t h e l i g h t
and heavy chains of myosin are t r a n s l a t e d on d i f f e r e n t mRNA's and
subsequently assembled t o form n a t i v e myosin (Sarkar and Cooke, 1970;
1971; Brivo and F l o r i n i , 1971). I n a d u l t s k e l e t a l muscle
Low e t, * ,a 1
t h e r e a l s o appears t o be no one-to-one coordination i n the synthesis
of myosin chains (Morkin 5 &., 1973). I n cardiac muscle, t h e t u r n over r a t e of heavy myosin chain w a s about twice t h a t of t h e light
myosin chain, however, turnover of t o t a l myosin p r o t e i n r e f l e c t e d t h e
-
heavy chain myosin a s it accounts f o r a t l e a s t 80$ of the t o t a l protein
(Wikman-Coffelt e t a l . , 1.973).
Localization of ribosomes i n t h e myofiber
and t h e i r p o t e r , t i a l r o l e
i n t h e s p e c i f i c i t y f o r myofibrillar and soluble protein synthesis
I n ty-pical c e l l s , t h e bulk of protein synthesis occurs on microsomal ribosomes. Nuclei and mitochondria a r e among other c e l l u l a r
components t h a t e x h i b i t p m t e i n synthesis. F l o r i n i (1964) reported
t h a t t h e r a t e of protein synthesis of muscle microscomes was much lower
than t h a t of l i v e r microsomes; however, muscle mitochondria were a s
a c t i v e a s l i v e r microsomes (McLean & Q., 1958). On f r a c t i o n a t i o n ,
t h e bulk of muscle RNA sediments in t h e myofibril and n u c l e i f r a c t i o n ,
unlike l i v e r where t h e highest l e v e l s of RNA a r e found i n the microsomes,
(Narayanana and Eapen, 1973; Hulsman, 1961). The extent of RNA
recovery i n t h e various f r a c t i o n s of muscle i s dependent upon the ionic
strength of t h e homogenizing buffer. Heywood e t a l . (1968) showed t h a t
t h e ribosome y i e l d from muscle could be v a s t l y improved by using high
ionic strength buffer, a s muscle polysomes coprecipitated w i t h myosin
during homogenization of t h e t i s s u e i n lower ionic strength buffers.
Zak e t a l . (1967) i s o l a t e d t h e RNA associated with myofibrils of chick
embryonic h e a r t . These workers concluded t h a t most of the RNA was
firmly bound t o t h e myofibrillar s t r u c t u r e and 85% of t h e RNA was i n
t h e form of ribosomal RNA and s h i l a r t o microsomal rRNA. Recent
electron-micrograph work has t e n t a t i v e l y i n d i c a t e d , t h a t these ribosomes
a r e associated with t h e myosin filaments (Larson e t a l . , 1969). Winnick
and Winnick (1960) could not demmstrate t h a t f i b r i l l a r proteins were
assembled on microsomal ribosomes. They suggested t h a t f i b r i l l a r
proteins may be synthesized i n p a r t by microsomes o r by a t o t a l l y
independent system. Narayanan and Eapen (1973a,b) have shown t h a t
i s o l a t e d myofibrillar ribosomes a r e a c t i v e i n protein synthesis and
a r e not dependent on c e l l sap f a c t o r s f o r t h e i r a c t i v i t y . They also
demonstrated t h a t myosin is assembled by myofibrillar rRNA and concluded
t h a t t h e r e i s extensive and independent protein synthesis by myofibrils,
with t h e myofibrillar ribosomes as t h e functional u n i t s . The usual
microsomal preparation from muscle (without high ionic strength buffers )
presumably produce nonmyofibrillar p r o t e i n s . The amount of rqyosin
synthesis i n such c e l l f r e e systems is usually small ( R . Bjerke, 1974,
Iowa S t a t e University--personal communication )
--
--
.
I n i t i a t i o n f a c t o r - 3 (IF-3) has been implicated a s an obligatory
component of mRNA binding t o t h e small ribosomal subunit (Vermeer e t a l . ,
1973; Weissbach and Brot, 1974) and it has been proposed t h a t IF3 plays
a r o l e i n messenger s e l e c t i o n and s p e c i f i c i t y of protein synthesis.
Thus, Heywood & &. (1974) and Thompson and Heywood (1974) have
proposed that IF3 governs a f i n e tuning mechanism f o r the synthesis of
m y o f i b r i l l a r and sarcoplasmic proteins i n musCle c e l l s . On the other
hand, t h e f a c t t h a t ribosomes a r e located i n s p e c i f i c s i t e s i n muscle
c e l l s w o u l d argue t h a t the c e l l u l a r location of ribosomes may a t l e a s t
play a r o l e i n t h e s p e c i f i c i t y of p r o t e i n synthesis. More recent work
253
i n r a t l i v e r has shown t h a t d i f f e r e n t populations of ribosomes a r e
segregated i n t h e c e l l r a t h e r than a s widely held t h a t extensive
exchange takes place among ribosomes within a c e l l (Arora and Robert,
1974). This c e l l u l a r s i t e suggestion has some support from t h e finding
(Srivastava and Chaudhary, 1969) that t h e ribosomes associated with t h e
nuclei-myofibril f r a c t i o n ( p r i m a r i l y involved i n s t r u c t u r a l p r o t e i n
s y n t h e s i s ) declined 5 f o l d from b i r t h t o old age, while t h e rRNA
associated with t h e supernatant, microsomes and mitochondria only
declined 2-2.5 f o l d over t h e same period. I n Later l i f e ( p o s t growth),
it would appear t h a t t h e synthesis of sarcoplasmic and s o l u b l e p r o t e i n s
(enzymes involved i n energy metabolism, e t c ) would be r e l a t i v e l y more
important than t h e synthesis of s t r u c t u r a l p r o t e i n s .
.
Muscle p r o t e i n turnover
The concept of a dynamic state of t i s s u e and t h e cycling and t u r n over of t i s s u e components w a s firmJ.y introduced by Schoenheimer (1942).
The process of p r o t e i n degradation i s q u a n t i t a t i v e l y almost as important
a s p r o t e i n synthesis (Swick and Song, 1974).
A k i n e t i c a n a l y s i s of t h e processes of p r o t e i n synthesis and
degradation has been made by Schinke (1970). He showed that synthesis
i s a zero-order process, e.g. not dependent on t h e concentration of
r e a c t a n t s , while t h e degradation r a t e i s a first order process, e.g.
dependent on t h e concentration o f t h e r e a c t a n t s . Work i n p r o t e i n
degradation h a s a l s o shown t h a t t h e d i f f e r e n t p r o t e i n s have t h e i r own
s p e c i f i c r a t e constant which appears t o be a property of t h e s p e c i f i c
p r o t e i n and i t s i n t e r a c t i o n with o t h e r c e l l u l a r f a c t o r s (Swick and
Song, 1974).
A major problem f o r a l l s t u d i e s on in vivo synthesis o r degradation
rates of p r o t e i n s with radioisotopes, e s p e c i a l l y a s i n g l e dose adminis t r a t i o n , is a proper analysis of t h e s p e c i f i c a c t i v i t y of t h e precursor
pool, and a l s o t h e recycling and r e u t i l i z a t i o n of l a b e l l e d amino a c i d s .
To overcome t h e l a b e l r e u t i l i z a t i o n problem, a v a r i e t y of approaches
have been developed t o i n s u r e removal of t h e l a b e l l e d amino a c i d from
t h e precursor pool a f t e r one cycle. Two common approaches t o overcome
r e l a b e l l i n g problems have been t o flood t h e system with t h e unlabelled
compound and/or feeding high p r o t e i n d i e t s o r t use a p r o t e i n precursor
such a s guanidino 14C l a b e l l e d a r g i n i n e o r Na$CO3
as alanine and glutamic
a c i d , which have a high metabolic degradation r a t e and, thus, a r e l e s s
extensively r e u t i l i z e d (Swick and Song, 1974; Schimke, 1970). The
approach of overloading with unlabelled amino acids (chase) appears t o
be somewhat undesirable a s t h e feeding of high p r o t e i n d i e t s o r s i n g l e
amino a c i d s may induce a physiological response t h a t w i l l i n t e r f e r e with
t h e desired r e s u l t s .
2 54
Most e a r l y work on protein degradation was done i n l i v e r and some
of t h e procedures used i n l i v e r a r e not applicable i n muscle. Thus,
w i t h s i n g l e i n j e c t i o n experiments it was shown t h a t l a b e l l e d a r g i n i n e
was extensively r e u t i l i z e d (Millward, 197Ck; Swick and Handa, 1956).
Labelled (li4-C)
glutamic a c i d appeared t o be a n excellent compound t o
study degradation i n muscle p r o t e i n and gave r e s u l t s similar t o l 4 C
carbonate i n j e c t i o n s (Swick and Song, 1974). When animals a r e fed a
p r o t e i n d e f i c i e n t d e t o r a r e i n a wasting s t a t e , t h e use of a s i n g l e
administration of l'&-glutamate w i l l not give v a l i d estimates o f p r o t e i n
turnover s i n c e t h e l a b e l was extensively recycled. Under t h e s e circumstances, cumbersome techniques of infusion o r perfusion become necessary
where t h e s p e c i f i c a c i d o r t h e l a b e l l e d species i n question i n t h e
soluble p o o l can be continuously monitored (Swick and Song, 1974).
The e f f e c t of s t a r v a t i o n , law p r o t e i n d i e t s , hormones and induced
muscle hypertrophy on s k e l e t a l mixed muscle p r o t e i n synthesis and
degradation has been s t u d i e d . Starvation o r feeding p r o t e i n f r e e d i e t s
decreased t h e r a t e of synthesis and increased t h e r a t e of breakdown of
1971). When
p r o t e i n s i n muscle of r a t s (Millward, 1970a; Young e t a1
r a t s were refed an adequate p r o t e i n d i e t , i n i t i a l l y t h e degradation of
muscle p r o t e i n s was stopped and synthesis of p r o t e i n s was increased,
b u t a f t e r a long period of refeeding, t h e mixed s k e l e t a l muscle p r o t e i n
degradation r a t e returned t o normal (Young e t a l . , 1971).
.,
I n i n j u r y induced hypertrophy of rat soleus muscle, t h e r e was
decreased catabolism and increased synthesis of mixed muscle p r o t e i n s
Furthermore, t h e degradation of t h e soluble ( sarco (Gcldberg, 1969a )
plasmic) p r o t e i n s decreased more markedly than t h a t of t h e m y o f i b r i l l a r
p r o t e i n s r e s u l t i n g i n a r e l a t i v e increase of sarcoplasmic p r o t e i n
a c c r e t i o n during i n j u r y induced compensatory growth (Goldberg, 1969a).
Growth hormone, however, increased p r o t e i n synthesis i n l e g s k e l e t a l
muscle without changing t h e p r o t e i n degradation r a t e (Goldberg, l g 9 a ) .
When rats were t r e a t e d w i t h cortisone, t h e r e w a s an increase i n
degradation of mixed p r o t e i n s of s k e l e t a l muscle (Goldberg, l969b).
.
During hypertrophy induced by muscle denervation of r a t diaphrams,
t h e r a t e of p r o t e i n synthesis i n i t i a l l y increased t o more than twice
t h e normal r a t e and then declined t o 5G$ of t h e normal r a t e , however,
t h e r a t e of mixed muscle p r o t e i n degradation a l s o increased t o more t h a n
twice t h e normal r a t e of s k e l e t a l muscle (Turner and Garlick, 1974).
The turnover r a t e constant as w e l l as h a l f - l i f e of p r o t e i n s i s (as
outlined above) dependent on t h e extent of l a b e l recycling. The more
recycling occurs during a study, t h e lower t h e degradation r a t e and t h e
longer t h e h a l f - l i f e . W8terlow and Stephen (1968) and Garlick (1969)
reported t h a t t h e average half - l i f e of mixed ,muscle p r o t e i n w a s 5-6
days. Half-lives f o r muscle sarcoplasmic and m y o f i b r i l l a r p r o t e i n s i n
normal r a t s were 3.6 and 15.6 days, r e s p e c t i v e l y (Millward, 1970b). I n
p i g s k e l e t a l muscle, t h e h a l f - l i v e s f o r mixed sarcoplasmic and mixed
m y o f i b r i l l a r p r o t e i n s were 9.4 and 16.4 days, r e s p e c t i v e l y (Perry, 1974).
255
The h a l f - l i f e for intramuscular connective t i s s u e proteins i n pigs was
20 days (Perry, 1974). The h a l f - l i v e s f o r r a t s k e l e t a l muscle f o r
p u r i f i e d myosin and a c t i n have been shown t o be i n t h e range of between
20-45 and 50-70 days, r e s p e c t i v e l y (Swick and Song, 1974).
The r o l e of developmental and other hormones on muscle p r o t e i n synthesis
The hormones which play a r o l e i n t h e development and growth of
muscle a r e growth hormone ( G H ) , i n s u l i n , androgens and thyroxine.
Although t h e s e hormones promote p r o t e i n synthesis mechanism, they a r e
not an obligatory component of t h e p r o t e i n synthesis mechanism. P r o t e i n
a c c r e t i o n during work induced hypertrophy has been found i n hypophysectomized rats (Goldberg, 1967) and Leathem and Koishi (1972) reported
t h a t GH o r i n s u l i n were not needed f m t h e r e p l e t i o n of t i s s u e p r o t e i n s .
The mecbnism(s) by which hormones stimulate p r o t e i n synthesis i s
not w e l l d e t a i l e d . The operon, r e g u l a t o r gene, repressor and derepressor
hypothesis of Jacob and Monod (1961) which predicted t h e existence of
a n unstable mRNA a s a mechanism f o r q u a l i t a t i v e and q u a n t i t a t i v e c o n t r o l
of p r o t e i n synthesis w a s applied t o hormonal regulation. Thus, it was
proposed t h a t hormones might a c t as a derepressor. However, i n c e l l s
of higher animals t h e regulation of p r o t e i n synthesis occurs a t many
s i t e s other than mRNA synthesis (Williams-Ashman and Reddi, 1972). Tata
( 1968) has proposed t h a t developmental hormones (and androgens ) promote
t i s s u e p r o t e i n synthesis by enhancing t h e synthesis of ribosomes ( p r o t e i n
synthesis machinery). Breuer and F l o r i n i (1965,1966) found t h a t t e s t o s terone increased t h e template a c t i v i t y of DNA i n chromatin and a l s o
p r o t e i n synthesis i n r a t s k e l e t a l muscle. Androgens did not, however,
promote t h e synthesis of any s p e c i f i c muscle p r o t e i n s ( F l o r i n i , 1970).
I n s u l i n promotes p r o t e i n synthesis by a mechanism independent of
i t s r o l e i n glucose metabolism (Manchester, 1972). The i n s u l i n e f f e c t
i n muscle i s e i t h e r r e l a t e d t o amino acid uptake (Goldstein and Reddy,
1970), but discounted by London (1973) and Manchester (1972), o r t o a
d i r e c t e f f e c t on t h e t r a n s l a t i o n (Manchester, 1972). The r o l e of
i n s u l i n i n muscle p r o t e i n s y n t h e s i s i s not limited t o DNA dependent RNA
synthesis (Woll, 1972) i n c o n t r a s t t o androgens and developmental
hormones. Growth hormone appears t o primarily influence t i s s u e p r o t e i n
synthesis by stimulating RNA synthesis (Korner, 1967; Manchester, 1970),
but others have shown t h a t GH enhances elongation (Kostyo and R i l l e m a ,
1971). The r o l e of thyroxine i n muscle p r o t e i n i s n o t c l e a r ; however,
decreased t h e r a t e of elongation i n r a t l i v e r (Ma'chews
,*,
_.
Overall developmental aspects, c e l l u l a r i t y , nucleic a c i d metabolism and
e f f i c i e n c y of p r o t e i n synthesis during muscle growth
The o v e r a l l growth p a t t e r n of s k e l e t a l muscle (Enesco and Puddy,
1%;
Chiakulus and Pauly, 1965) has been o u t l i n e d from s t u d i e s with
rats and mice. During e a r l y p o s t n a t a l growth ( p e r i n a t a l ) t h e r e i s a
3 f o l d increase i n t o t a l DNA and n u c l e i i n muscle, whereas muscle mass
increases approximately 4-5 f o l d . This period is generally considered
one of hyperplasia and some hypertrophy. I n t h e young a d u l t stage
t h e r e i s only a f i f t y percent increase i n n u c l e i and DNA content while
muscle mass increases about 3 f o l d . This period i s g e n e r a l l y c a l l e d
hypertrophy. Throughout t h i s period ( b i r t h t o young a d u l t s t a g e ) t h e
number of myofibers per muscle, hmever, remain constant. The increase
i n DNA i n muscle during growth has been a t t r i b u t e d t o mitosis i n
u n d i f f e r e n t i a t e d myoblasts, s a t e l l i t e c e l l s and o t h e r c e l l s (Moss and
Leblond, 1971; Mauro, 1961), while it i s generally accepted t h a t
myofiber numbers a r e e s t a b l i s h e d p r i o r t o b i r t h o r during t h e p e r i n a t a l
period and growth i n muscle mass occurs through increases i n i n d i v i d u a l
myofibers (Young, 1970; Goldspink, 1972).
Changes i n muscle nucleic a c i d and p r o t e i n content o r concentrations
have a l s o been studied w i t h a number of meat producing animals. I n both
pigs (Robinson, 1969) and sheep (Johns and Bergen, 1973) t o t a l DNA, RNA
and p r o t e i n increase during muscle growth. Nucleic acid concentrations,
however, decline continuously from b b t h t o a f i n a l l e v e l due t o t h e
rapid increzses of o t h e r c e l l u l a r components ( e s p e c i a l l y p r o t e i n ) of
muscle. I n normal growth, i n pigs and sheep, changes i n RNA and DNA
were p a r a l l e l , while t h e protein/DNA o r protein/RNA r a t i o s increased
markedly (Robinson, 1969; Johns and Bergen, 1973). There a l s o appears
t o be l i t t l e change i n protein/DNA r a t i o (an i n d i c a t i o n o f c e l l s i z e )
during l a t e r growth s t a g e s . Cheek e t a l . (1971) have suggested t h a t
each nucleus controls a f i n i t e mass of cytoplasm. I n "double muscle"
c a t t l e , muscle protein/DNA and RNA/DNA r a t i o s were similar t o normal
genotype c a t t l e (Ashmore and Robinson, 1969). Topel (1971)reported
t h a t a l e a n s t r a i n of pigs had c o n s i s t a n t l y higher RNA/DNA and p r o t e i n /
DNA r a t i o s than a f a t s t r a i n of p i g s (Poland China). Powel and Aberle
(1975) showed, however, t h a t o v e r a l l , i n heavy and l i g h t muscled Duroc
pigs, protein/DNA and RNA/DNA r a t i o s d i f f e r e d l i t t l e . I n summary then,
during normal growth (adolescence), t h e DNA and RNA concentrations i n
muscle d e c l i n e markedly and then s t a b i l i z e i n t h e a d u l t , while t h e
protein/HNA reaches i t s p l a t e a u value. It appears t h a t increases i n
muscle mass a r e e n e r a l l y accwpanied by a proportional increase i n
DNA (e.g. n u c l e i
An induced p r o l i f e r a t i o n of ruuscle n u c l e i during
p o s t n a t a l growth ( i.e
enhancement of s a t e l l i t e c e l l DNA production)
may thus be an approach t o increase t o t a l muscling in animals.
--
7 . .,
This general p a t t e r n of constant c e l l s i z e (protein/DNA r a t i o )
can be modified by n u t r i t i o n a l s t r e s s in animals, Thus, when sheep
were f e d a low (7%)p r o t e i n r a t i o n during a 60 day, post weaning,
feeding t r i a l , muscle RNA/DNA r a t i o s declined b u t protein/DNA r a t i o s
were a c t u a l l y somewhat increased (Johns and Bergen, 1973). While t h e
l a c k of s u b s t r a t e (amino a c i d s ) f o r p r o t e i n s y n t h e s i s tends t o depress
t h e production of r R N A (WaMemaCher, 1972) concomitantly t h e p r o t e i n
degradation i s depressed (Millward e t a1 1974) possibly accounting
f o r an e l e v a t i o n i n protein/DNA r a t i o s . When rat pups were assigned
i n groups of 3, 8 o r 16 per dam o r malnourished during e a r l y p o s t n a t a l
growth, t h e rats having t h e lowest w e i t gain had t h e l a r g e s t p r o t e i n /
DNA r a t i o s i n muscle (Vastus l a t e r a l i s (Stewart, 1974). These animals,
.,
therefore, exhibited an inverse r e l a t i o n s h i p between muscle s i z e
(protein/DNA r a t i o , Cheek e t al., 1968) and l i v e weight gain. The
author (Stewart, 1974) concluded t h a t t h e l a r g e r t h e c e l l s i z e i n
muscle t i s s u e a t a p a r t i c u l a r body weight, t h e lower t h e p o t e n t i a l f o r
f u r t h e r body growth.
--
Efficiency of protein synthesis can be defined a s t h e p o l e s of
amino acids incorporated per mg polysomal RNA ( r R N A ) per u n i t time.
From a long term regulation view, t h e l e v e l of t i s s u e r R N A i s t h e major
f a c t o r influencing t h e r a t e of protein synthesis (IJannemacher, 1972)
Millward e t a l . (1973) and Henshaw e t a l . (1971) showed a d i r e c t r e l a t i o n s h i p between p r o t e i n synthesis and t i s s u e polysomal RNA content and
a highly s i g n i f i c a n t ( p o s i t i v e ) r e l a t i o n s h i p between growth r a t e s i n
r a t s and t h e e f f i c i e n c y of protein synthesis in muscle and l i v e r . The
mechanism f o r t h i s improved r a t e of amino acid incorporation per u n i t
polysomal RNA i s not c l e a r , presumably it i s mediated by soluble c e l l
sap f a c t o r s , hormonal f a c t w s and other f a c t o r s .
--
.
L-
I n past work, t h e number of ribosomes a c t i v e l y involved in protein
synthesis have been measured with polysomal p r o f i l e s , polysomal rRNA/
t o t a l RNA r a t i o s o r a s t h e r a t i o of subunit RNA t o polysomal RNA. Thus,
increased protein synthesis w a s usually ascribed t o an increased number
of polysomes. All polysomal ribosomes may not be a c t i v e i n protein
synthesis, however (Sussman, 1970). To c l a r i f y t h i s problem, Nwagwu
and N a n a (1974) studied polysomal p r o f i l e s and I t a c t i v e ribosomes" i n
11, 14 and 17 day old embryonic chick l e g muscles. During t h i s period
t h e r e i s a 3 f o l d ( a t l e a s t ) increase i n muscle p r o t e i n synthesis r a t e .
Throughout t h e study, 9% of t h e ribosomes were present i n polysomes and,
thus, t h e amount df polysomal RNA cannot account f o r t h e enhanced r a t e
of p r o t e i n synthesis (Nwagwu and Nana, 1974). These workers estimated
t h e number of t R N A per ribossmes i n the polysome. It was reasoned t h a t
a ribosome carrying t w o tRNA (one each a t t h e P and t h e A s i t e ) would
be a c t i v e . The number of "active" ribosomes were s i m i l a r f o r 11, 14 and
17 day embryonic chick muscle. Muscle p r o t e o l y t i c a c t i v i t y was low and
a l s o not d i f f e r e n t f w t h e three age groups. These workers (Mwagwu and
Nana, 1974) therefore concluded t h a t t h e increased r a t e of protein
accretion ( f r m 11 t o 17 days of age) must be r e l a t e d t o an increase
i n t h e e f f i c i e n c y of protein synthesis by t h e ribosomes. Bergen (1974)
depressed growth (weight gain) i n r a t s by r e s t r i c t i n g feed intake of an
optimal d i e t t o 50% (dry weight) of a control ad l i b group, but could
not demonstrate differences in muscle protein synthesis e f f i c i e n c y
r e l a t e d t o o v e r a l l growth. It was s h m , however, t h a t t o t a l carcass
protein accretion was s i m i l a r between t h e two groups of r a t s and the
e x t r a w e i g h t gain i n t h e c o n t r o l animals was mostly f a t (Bergen,
unpublished).
--
Problems i n studying t h e r o l e of n u t r i t i o n and hormones i n muscle
protein synthesis mechanism
Great e f f o r t has been expanded by many workers on t h e r e l a t i o n s h i p
of hormonal and n u t r i t i o n a l changes with p r o t e i n synthesis i n the
2 58
muscle ( s e e Bergen, 1974; Trenkle, 1974;and Young, 1974). Although it
i s c l e a r t h a t lack of s u b s t r a t e ( p r o t e i n or energy) w i l l depress t h e
l e v e l and a c t i v i t y of p r o t e i n synthesis machinery, t h e r e i s l i t t l e
i n f o r m t i o n about t h e r a t e l i m i t i n g s t e p during such a physiological
s t a t e ( s ) . The proposed a c t i o n of t h e various hormones a r e o f t e n very
speculative, derived from i n v i t r o work and a r e probably o f t e n not
applicable t o t h e i n vivo s i t u a t i o n . A good example of such a problem
i s t h e inverse r e l a t i o n s h i p between growth r a t e i n s t e e r s and serum GH
l e v e l s (Grigsby, 1973)
-.
To study o v e r a l l a s p e c t s of muscle p r o t e i n synthesis r a t e s ( o t h e r
than an assessment of net a c c r e t i o n during a feeding period) e i t h e r 5
v i t r o o r & vivo, procedures using r a d i a c t i v e l a b e l l e d s u b s t r a t e s (amino
a c i d s ) have been employed.
Although t h e i n vivo approach should y i e l d generally u s e f u l r e s u l t s ,
t o o o f t e n research r e p o r t s appears t h a t use such naive approaches t h a t
t h e r e s u l t s a r e u s e l e s s . Recently, Hsueh e t a l . (1975)reported t h a t
muscle p r o t e i n synthesis r a t e was s i m i l a r f o r r a t s f e d a poor q u a l i t y
p r o t e i n o r high q u a l i t y protein d i e t , although t h e r e w a s a t h r e e f o l d
d i f f e r e n c e i n weight gain. P r o t e i n s y n t h e s i s w a s measured as CPM
incorporated per 100 mg p r o t e i n i s o l a t e d . This approach i s unfortunately
a l l t o o common, b u t t o t a l l y inappropriate. To study p r o t e i n synthesis
i n vivo ( i n any given organ), t h e s p e c i f i c a c t i v i t y 3f t h e precursor
pool during t h e time course of t h e incorporation period must be described.
T h i s problem i s even more important i f only a t r a c e r dose (no cold
c a r r i e r ) i s administered. There i s considerable disagreement as t o
t h e confines of t h e "precursor pool"; however, t h e s p e c i f i c a c t i v i t y
of t h e l a b e l l e d amino acid i n t h e c e l l u l a r tEWA, nascent peptides
chains ( I l a n and Singer, 1975) o r i n t h e t o t a l acid soluble t i s s u e
e x t r a c t (Fern and Garlick, 1974) can be measured. S p e c i f i c a c t i v i t y
determinations i n acyl-tmA and nascent chains a r e q u i t e involved and
not o f t e n attempted. The t o t a l t i s s u e pool approach does present a
useful estimate t h a t i s g e n e r a l l y not t o o d i f f e r e n t from t h e " r e a l "
precursor pool (Khairallah and Mortimore, 1975).
-_I_
7
-
The e x t e n t of t h e d e s c r i p t i o n of t h e l a b e l l i n g p a t t e r n of t h e
precursor pool s p e c i f i c a c t i v i t y ( s e e t h e following f o r methods and
t h e o r e t i c a l considerations of s p e c i f i c a c t i v i t i e s : Garlick and Millward,
1972; Henshaw e t a l . , 1971; Airhart e t al., 1974; Garlick
g . , 1973;
Fern and Galick, 1973, 1974; I l a n and Singer, 1975; Venrooij e t a l . ,
1974) depends on t h e questions posed by t h e i n v e s t i g a t o r . For s t u d i e s
on t h e e f f e c t of a given treatment o r v a r i a b l e on s e v e r a l aspects of
p r o t e i n synthesis (e.g. increase, no change o r decrease) r a t e , it need
only be shown t h a t t h e product's (e.@;.newly synthesized o r completed
muscle p r o t e i n s ) r a d i o a c t i v e amino a c i d content i s not merely a function
of s p e c i f i c a c t i v i t y changes in t h e t i s s u e (precursor) f r e e amino acid
pool. For more q u a n t i t a t i v e work and c a l c u l a t i o n s of t h e e f f i c i e n c y of
p r o t e i n synthesis, t h e s p e c i f i c a c t i v i t i e s of t h e t i s s u e (precursor)
f r e e amino a c i d pool must be determined. A t t h e same time, a s a good
precaution, t h e o v e r a l l r o l e of given experimental v a r i a b l e s on
precursor pools (amino a c i d concentrations ) should be s t u d i e d .
I
-
The i n vitro, c e l l f r e e p r o t e i n synthesis systems avoid t h e
p r e c u r s o r p o o l problems, but a host of new problems a r e introduced.
The r o u t i n e i s o l a t i o n from muscle of microsomal systems (ribosomes )
and soluble f a c t o r s necessary f o r c e l l f r e e p r o t e i n synthesis i s q u i t e
d i f f i c u l t . Low i o n i c s t r e n g t h b u f f e r s only remove a s m a l l f r a c t i o n of
t h e RNA i n muscle, increasing t h e i o n i c s t r e n g t h improves t h e y i e l d
markedly; b u t nevertheless l e s s t h a n h a l f of t h e c e l l u l a r RNA i s e x t r a c t e d .
i n v i t r o c e l l free muscle systems i s generally
The a c t i v i t y of homologms -q u i t e l o w (less than 3$ of t h e i n vivo r a t e ) prompting i n v e s t i g a t o r s t o
use m l y muscle ribosomes and l i v e r soluble f a c t o r s and precharged t R N A .
The microsomes removed from muscle a t low i o n i c s t r e n g t h are not t y p i c a l
a s they make f e w f i b r i l l a r p r o t e i n s . The polysomes extracted a t higher
i o n i c s t r e n g t h (Heywood e t a l . , 1968) most l i k e l y a r e devoid of i n i t i a t i o n
f a c t o r s ( e .g. s a l t washed ribosomes, Lucas-Lenard and Lipmann, 1971)
and t h e s e systems can only f i n i s h t h e already s t a r t e d nascent peptide
chains. Thus, an i n v i t r o approach t o assess n u t r i t i o n a l o r hormonal
modulation of p r o t e i n synthesis i n muscle i s n o t adequate. Carefully,
r e c o n s t i t u t e d muscle c e l l free p r o t e i n synthesis systems a r e , however,
very u s e f u l i n t h e study of t h e r o l e of IF, elongation f a c t o r s , tRNA,
t h e r o l e of ribosomal proteins, etc., in muscle p r o t e i n synthesis; but
t h e s e systems seldom r e f l e c t any physiological circumstances which a r e
of primary i n t e r e s t t o t h e meat and animal s c i e n t i s t .
--
--
Mechanisms of p r o t e i n synthesis ( t r a n s l a t i o n ) i n growing meat type
animals ( a s w e l l a s laboratory rodents) w i l l not be studied e f f e c t i v e l y
u n t i l new experimental approaches have been evolved. During n u t r i t i o n a l
and hormonal challenges, i n i t i a t i o n , elongation and possibly termination
may become rate l i m i t i n g ( c o n t r o l p o i n t ) t o t r a n s l a t i o n . There a r e now
no e f f e c t i v e procedures t o study t h e s e processes without destroying the
physiological and u l t r a s t r u c t u r a l milieu of t h e muscle system. Some of
t h e newer approaches i n studying i n i t i a t i o n r e a c t i o n s ( e .g N t e r m i n a l
v s . i n t e r n a l methionine uptake; Oleinick, 1975), a d i r e c t a s s a y of I F
concentrations i n crude muscle ribosome e x t r a c t s (Lubsen and Davis,
1974), o r use of s p e c i f i c i n i t i a t i o n i n h i b i t o r s (Bergen, 1974a) may be
of some value i n studying i n i t i a t i o n i n muscle systems. It may w e l l be
t h a t i n s t e a d of using w e l l described c e l l f r e e p r o t e i n synthesis
incubation systems, t h e k i n e t i c synthesis machinery ( e .g myosin, a c t i n ,
s p e c i f i c enzymes) i n i n v i t r o crude muscle homogenates o r i n i n vivo
s t u d i e s w i l l become a more appropriate t o o l t o study t h e e f f e c t of
n u t r i t i o n a l changes, hormone s e c r e t i o n s and growth r a t e modifications
on t r a n s l a t i o n and p r o t e i n deposition in muscle.
.
.
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T . R . Dutson: I t h i n k f i r s t w e ' l l c a l l for some d i s c u s s i m between
t h e panel members. I knar some of you have some i n t e r e s t i n g questions
you would l i k e t o a s k each o t h e r about your p a r t i c u l a r t o p i c s . SD, I
t h i n k w e ' l l s t a r t out with i n t e r d i s c u s s i o n here and t h i s might s t i m u l a t e
d i s c u s s i o n from t h e audience. A t t h e p o i n t t h a t people i n t h e audience
have q u e s t i o n s , if you would just r a i s e your hands, we can have a broad
d i s c u s s i o n back and f o r t h .
C . E . Allen: Ron, I ' d l i k e t o a s k you, i s t h e r e any p r o l i f e r a t i o n
due t o t h e i n s u l i n o r o t h e r hormones a t t h i s point?
R. E . Allen: We have looked a t t h e e f f e c t s of various l e v e l s of
i n s u l i n on c e l l p r o l i f e r a t i o n with increasing l e v e l s of i n s u l i n . The
only problem is, t o g e t t h i s increase, you're working with l e v e l s of
i n s u l i n which a r e two t o t h r e e orders of magnitude g r e a t e r t h a n what
you would f i n d p h y s i o l o g i c a l l y
.
W. G . Bergen: On t h e question of turnover, it has been suggested
that energy i s required t o keep t h e calcium under c o n t r o l .
. .
W R Dayton: There i s obviously an energy requirement of t h e SR
f o r t a k i n g u p calcium and s o it i s p o s s i b l e that t h e r e i s energy involved.
R . H. F i t t s :
W h a t i s t h e i d e a l pH f o r t h e lysosomal enzymes?
V I . R . Dayton:
It v a r i e s with t h e d i f f e r e n t enzymes but I would
guess, g e n e r a l l y speaking, i t ' s down c e r t a i n l y i n t h e range below pH
5 t o 5 1/2, down t o even 4 i n some c a s e s . There a r e r e p o r t s of some
lysosomal enzymes that a r e a c t i v e a t higher pH's b u t I d o n ' t t h i n k t h e y
a r e extensive
.
R. H. F i t t s : I n c e r t a i n types of e x e r c i s e s , t h e r e can be a lower
i n t r a c e l l u l a r pH, perhaps down as low as 6 b u t nothing n e a r l y t h e
range t h a t you a r e t a l k i n g about.
W . R . Dayton:
I guess what I would l i k e t o emphasize i s that t h e
reason that I dwelled on t h e lysDsoiml t h i n g s o much i s t h a t I t h i n k
maybe i n t h e past we looked at"1ysosomes because t h e y were t h e r e ,
because w e knew t h e y were t h e r e , and w e may have looked t o o hard f o r
t h e r o l e of cathepsins in that maybe t h e y a r e a l i t t l e t o o g e n e r a l t o
do t h e kinds of t h i n g s that we s e e .
Question: There seems t o be considerable controversy as t o what
is t h e normal i n t r a c e l l u l a r pH of muscle c e l l s .
R . H. F i t t s : There i s a r e a l controversy i n that a r e a now and t h e
main reason, I t h i n k , i s due t o t h e f a c t that t h e pH-sensitive e l e c t r d e s
that people a r e u s i n g , some of them a r e not designed properly i n t h e
sense that t h e y are r e a l l y made sc) that yau a r e r e a l l y measuring e x t r a c e l l u l a r pH, t h e y a r e r e a l l y made f o r e x t r a c e l l u l a r a c t i o n .
T . R. Dutson: I would l i k e t o a s k B i l l i f you have looked a t t h e
r e l a t i o n s h i p between t h e a c t i v i t y of t h e CAF f a c t o r on t h e various
m y o f i b r i l l a r p r o t e i n s and t h e r a t e D f p r o t e i n turnover of t h e s e p r o t e i n s ;
i n other words, i s t h e r e a c o r r e l a t i o n between t h e r a t e of C A F a c t i v i t y
on t h e s e p r o t e i n s and t h e i r r a t e of turnover?
W . R . Dayton: We have none but t h e r e a r e s e v e r a l s t u d i e s t h a t have
indicated t h e r e l a t i v e r a t e of turnover of m y o f i b r i l l a r p r o t e i n s r e l a t i v e
t o each o t h e r and i n most cases troponin and tropomyosin a r e found t o
t u r n over more r a p i d l y than a c t i n and myosin. I d o n ' t mean here t o
imply that CAF does a l l of t h e turnover of m y o f i b r i l l a r p r o t e i n s , b u t
t h e a c t i v i t y of CAF on m y o f i b r i l l a r p r o t e i n s i s c o n s i s t e n t with d a t a
that has been published t o d a t e 3n t h e r e l a t i v e r a t e of turnover of
these proteins.
Question: Do you t h i n k that CAF can remain a c t i v e postmortem, and
how could we induce it t o be a c t i v e postmmtem?
. .
W R Dayton: Well, we have, but I want t o make it very c l e a r t h a t
t h i s work was done by Dennis Olson, another graduate s t u d e n t and not by
me; b u t , we have looked a t t h e p o s s i b l e e f f e c t s of CAF postmortem using
SDS g e l s on myofibrils and t h e only e f f e c t w e can f i n d on m y o f i b r i l l a r
p r o t e i n s postmortem a r e those which we can expect t o be caused by t h e
CAF a c t i v i t y . A s far as inducing t h e a c t i v i t y of t h e enzyme postmwtem,
r i g h t now, probably your guess i s as good as mine; we a r e thinking about
maybe ways of a c c e l e r a t i n g calcium r e l e a s e , of c o n t r o l l i n g pH d e c l i n e
and t h i n g s like t h i s t h a t w i l l optimize t h e a c t i v i t y of t h e enzyme.
But, we a r e now t a l k i n g about t h i n g s that mechanistically would be
p r e t t y d i f f i c u l t t o do and t h i s c e r t a i n l y r e q u i r e s some more study.
T . R. Dutson: B i l l , I have a question i n r e l a t i o n t o t h i s ; w h a t
i s t h e optimum temperature f o r t h i s enzyme?
W . R. Dayton:
In v i t r o i n i t i a l r a t e s of t h i s enzyme a r e m r e
r a p i d a t 3 7 O C t h a n a t any o t h e r temperature; h m e v e r , t h e enzyme is
f a i r l y u n s t a b l e a t 3 7 O C , it apparently hydrolyzes, i n f a c t , w e have
good evidence that it does. However, we d o n ' t r e a l l y know whether
that's a problem i n vivo.
T . R. Dutson: In t h i s l i g h t then, i f you could have a high enough
calcium concentration around t h i s enzyme a t a high enough temperature
t h e n postmortem degradation of t h e s e p r o t e i n s would t a k e place v e r y
r a p i d l y . Is that r i g h t ?
W
. R . Dayton:
Yes.
T. R. Dutson: If y ~ would
u
hold t h e temperature high and i f t h e
amount of calcium t h e r e w a s h i g h enough t o s t i m u l a t e t h i s enzyme a c t i v i t y
it would go a t a v e r y r a p i d r a t e .
268
!J. 3. Dayton:
This has a c t u a l l y been done by Dennis Olson: keeping
t h e muscle a t a higher temperature and t h e n using SDS g e l s as a marker
of t h e degradation, he found t h a t , D r Goll, c o r r e c t me i f I ' m wrong,
he found t h e same kind of changes happen i n maybe 2 days which would be
expected t o happen i n t h r e e days normally. However, it i s not c e r t a i n
nov as t o whether it i s CAF o r not causing t h i s ; I guess it c m l d be
any enzyme.
.
T . R . Dutson: Possibly it could be t h e combination t h a t you
mentioned e a r l i e r , CAF s t a r t i n g t h e process and other enzymes probably
f i n i s h i n g t h e degradation.
D. E . G o l l : I n terms of postmortem muscle i n s o f a r as we can
d i s t i n g u i s h t h i n g s on SDS g e l , everything we see happening i n postmortem
muscle on SDS g e l s , we can a t t r i b u t e t o CAF.
I presume from what has been s a i d t h e n t h a t , f o r
C . E . Allen:
example, i n PSE muscle a l l of the conditions would be very favorable
f o r an increase i n CAF a c t i v i t y ; such as maintaining a high postmortem
temperature, i n c r e a s e i n calcium o r l x s i n SR a c t i v i t y and higher o r
elevated temperature.
W . R . Dayton: The only problem would be t h e r o l e of pH. If you
g e t t h e p H r a p i d l y down arrund 5.5 or i n that range, t h e a c t i v i t y of
CAF would be decrkased.
R . H. F i t t s : Have you looked a t any models which achieve high
calcium concentrations more r a p i d l y , such as some dystrophies where
you have problems with t h e SR where t h e y don't t a k e up t h e calcium.
I wondered if t h e a c t i v i t y of CAF i s d i f f e r e n t i n t h e s e models.
W. R . Dayton: There a r e s e v e r a l people i n our l a b working on t h i s
now, on dystrophic muscle a t t h e present time and t h e y do f i n d a n
increase i n CAF a c t i v i t y (we are speaking of s p e c i f i c a c t i v i t y i n
crude p r e p a r a t i o n s ) . B u t I d o n ' t b e l i e v e there i s any change i n t h e
calcium requirement of C A F i n t h e dystrophic muscle. We d o n ' t r e a l l y
know w h a t t h e involvement of calcium i s , in other words, t h e f a c t t h a t
CAF a c t i v i t y i s increased does not n e c e s s a r i l y have anything t o do w i t h
t h e e f f e c t of calcium on t h e enzyme. But t h e f a c t t h a t t h e SR does
appear t o be impaired i n dystrophic muscle would t e n d t o f a l l i n l i n e
with t h e f a c t t h a t CAF does r e q u i r e higher calcium l e v e l s t h a n we t h i n k
tends t o be f r e e i n muscle c e l l s .
T . R . Dutson: I ' d l i k e t o g e t back t o t h e s u b j e c t of e x e r c i s e and
t h e d i f f e r e n c e s i n l e a n body mass. Bob, you mentioned t h a t t h e r e ' s a
decrease i n u t i l i z a t i o n of carbohydrate s t o r e s , glycogen, e t c , and
t h e r e f o r e , t h e muscle must use f a t t o increas? t h e amount of energy
and, of course, you showed t h e oxidation system increasing. However,
you mentioned t h e r e weren't any changes i n t h e muscle weight t o body
weight r a t i o . How do you account f o r t h i s when t h e y must use up f a t ;
i s it just t h e a m m t that's t h e r e due t o d i e t or do t h e y have t o use
up body s t o r e s of f a t ?
.
R. H . F i t t s : There's a good d e a l of work done a t t h e University
of Chicago studying t h e e f f e c t of exercise on c e l l numbers and c e l l
s i z e . S t a r t i n g t r a i n i n g e a r l y i n l i f e , they found that i f y3u exercise
e a r l y i n l i f e , it reduces t h e numbers of f a t c e l l s that w i l l u l t i m a t e l y
reach a c e r t a i n s i z e and t h a t even i f you terminate exercise, they w i l l
remain decreased, where on t h e other hand, i f y m reduce c e l l s i z e just
by r e s t r i c t i n g t h e d i e t apparently t h e a n i m l w i l l c3me back up t o
normal. It would seem t o be a d i f f e r e n t type of mechanism going on
t h e r e . I t h i n k it has been observed that c e r t a i n types of exercise
s t r e s s such as overload or weight l i f t i n g appear t o be a b l e t o increase
myofibrillar s i z e . It has been reported that t h e r e a r e increases i n
numbers of f i b e r s due t o running caused by s p l i t t i n g of f i b e r s , e t c .
I don't know whether t h a t ' s f a c t or a r t e f a c t a t t h i s stage of t h e game.
TiJe know that t h e r e i s no change i n muscle t o body weight r a t i o s .
D. E . Goll: The husband D f a technician working i n our l a b
bicycles a l D t and he claims t h e f o l k l o r e on b i c y c l e racing has it
t h a t about 4 days before t h e r a c e i f you w i l l f a s t a f e w days then
load up on carbohydrates you w i l l a p p r e n t l y build up glycogen.
R . H. F i t t s : The idea i s a week before t h e race, you go out and
run long runs and d e p l e t e your muscles of glycogen s t o r e s when you go
on a low carbohydrate d i e t t o f u r t h e r maintain t h i s . Then 3 days before
t h e race you s t a r t overloading, e a t i n g not n e c e s s a r i l y more t o t a l
c a l o r i e s but more t o t a l carbohydrates. People make t h e mistake of
e a t i n g mx-e t o t a l c a l o r i e s , l i k e e a t i n g 5 loaves of bread; t h e y invari a b l y have high muscle glycogen with a tremendous stomach ache! This
i s a p r a c t i c a l u t i l i z e d technique, and it does work; it increases t h e
muscle glyccgen 3 and 4 f o l d over normal l e v e l s .
F . B. Shorland: There seems t o be one t h i n g t h a t wasn't discussed
l a t e l y : it i s maybe a l i t t l e off t h e beat i n terms of t h i s meat
discussion, but I do t h i n k t h e study of meat i s a r a t h e r wonderful
t h i n g because it does bring us back t o t h e question of how t h e s e t h i n g s
e f f e c t humans. So t h e t o t a l e f f e c t on hurnans i s q u i t e g r e a t through
t h e study of meat. There i s one point, I don't know whether i t ' s been
worked on a t a l l , b u t does exercise promote longevity? The o t h e r t h i n g
that wasn't discussed is t h a t w e keep on l o s i n g calcium as we g e t older
and we go on l o s i n g protein, p a r t i c u l a r l y i n rnzles, which is r a t h e r
sad. But I t h i n k it has been indicated here that i f you kept on
exercising, you might b r i n g back a l i t t l e of t h e p r o t e i n , s o it would
be worthwhile t o keep everybody moving. W h a t I would l i k e t o know i s ,
t h e r e any r e l a t i o n s h i p t h a t anybody h e r e can see, which i s t h e premise
of t h e question, namely, i f you go on l o s i n g calcium is t h e r e any
connection t o t h i s and t h e reason why you go on losing protein? Anybody
c a r e t o comment on t h a t ?
D. E . Goll:
Is t h e calcium f r o m t h e bone o r t h e muscle?
F. B. Shorland: The l o s s i s probably from t h e bone, t h e skeleton.
Actually, I would l i k e t o see i f you can g e t anywhere on t h e lcngevity
one and i t s r e l a t i o n s h i p t o e x e r c i s e .
C . E. Allen: Well, i n that regard, it i s w e l l documented t h a t
overweight individuals have a s h o r t e r h a l f l i f e . When you g e t out t o
65 o r 70 years of age, many of t h e obese individuals have already died
and that t h e l o s s e s in lean body mass may be i n f a c t more dramatic i f
w e could keep them a l i v e and measure t h a t decrease, say between 50 and
70
R . H. F i t t s : I t h i n k t h e r e ' s no doubt i n my mind t h a t exercise
t r a i n i n g increases ones f e e l i n g of well-being through changes t h a t take
place i n t h e h e a r t and o t h e r muscles. Although t h e r e a r e no r e a l good
epidemiological s t u d i e s over time, and I might add they a r e very
d i f f i c u l t t o c o n t r o l due t o d i e t a r y i n t e r a c t i o n s , etc., s o it i s very
d i f f i c u l t t o study, but I w i l l say t h i s , d e f i n i t e l y t h e q u a l i t y of l i f e
o r t h e f e e l i n g of well-being is enhanced, b u t as far as longevity; I ' m
not sure you're going t o g e t d e f i n i t e answers on t h a t .
.
C E . Allen:
I might add t o w h a t Bob has s a i d , I t h i n k he made a
statement i n h i s p r e s e n t a t i o n about t h e a c t i v i t y of these animals that
were on longer term exercises. Currently, I have a student who i s
doing a study and he has some r a t s t h a t a r e exercised f o r d i f f e r e n t
periods of time and it i s very f a s c i n a t i n g t o go down t h e r e and, i f you
d i d n ' t know anything about t h e periods of time t h a t t h e s e rats were
being exercised, t h e one t h i n g you would note, just observing t h e
cages, i s t h a t those rats that are exercising f o r longer periods of
time, u s u a l l y when they g e t back i n t h e cages, are s t i l l doing something
and t h e statement that Bob made about t h e males having a lower body
weight than t h e controls which a r e unexercised i s also t r u e here. If
you just watch f o r a c t i v i t y , you can imagine why t h e y m i g h t be of a
lower body weight, and a d d i t i o n a l l y , t h e y appear t o have l e s s f a t on
t h e body.
T. R . Dutson: I'd l i k e t o a s k a question of B i l l Dayton and
Werner Bergen a l s o might l i k e t o coment on t h i s . Getting back t o t h e
p r o t e i n synthesis-degradation i n t e r a c t i o n and t h e n e t amount of p r o t e i n
synthesis due t o e i t h e r an increase o r decrease of e i t h e r one of t h e s e ,
would you c a r e t o speculate on some of t h e mechanisms by which t h i s
p r o t e i n synthesis could be increased. I t h i n k we would l i k e t o have
some s o r t of hypothesis come out of t h i s as t o w h a t t h e p o s s i b i l i t i e s
are t o increase t h e amount of proteins s y n t h e s i s . hhybe we need t o
decrease degradation, maybe we need t o increase s y n t h e s i s . Is t h i s
possible, and, if s o , how?
W. R . Dayton: I haven't r e a l l y been i n t o protein synthesis very
much; as far as p r o t e i n degradation goes, I guess t o be honest about
it, I would have t o say I don't knot: and I doubt whether anyone e l s e
does know what t h e e f f e c t of p r o t e i n degradation i s on t h e growing
animal. I suppose it could be something signi,ficant because t h e
capacity t o degrade p m t e i n is d e f i n i t e l y t h e r e ; but f o r some reason,
t h i s capacity i s completely shut off or n e a r l y shut off under normal
circumstances. I t h i n k it would be of i n t e r e s t t o f i n d out exactly
w h a t t h e mechanism is that controls t h e r a t e of p r o t e i n degradation
and whether the rate I s high or low. However, as f a r as p u t t i n g any
q u a n t i t a t i v e value on w h a t percent of growth or n e t p r o t e i n s y n t h e s i s
is due t o degradation, I don't t h i n k we can s a y . Another t h i n g we
might t h i n k about t o o , g e t t i n g away from m y o f i b r i l l a r p r o t e i n s , i s
t h a t degradation not only happens t o m y D f i b r i l l a r p r o t e i n s but it happens
t o p r o t e i n s of t h e lysosomes, it happens t o t h e synthetases and t o t h e
charging enzymes. So, you are not j u s t t a l k i n g about m y o f i b r i l l a r
p r o t e i n s b u t what happens t o t h e 70 some-odd p r o t e i n s i n t h e lysosomes
that m i g h t g e t degraded c)r synthesized. A l s o , some of t h e i n i t i a t i o n
f a c t o r s a r e involved i n p r e f e r e n t i a l s y n t h e s i s or degradation and t h e s e
are f a c t o r s that i m p a r t s p e c i f i c i t y t o t r a n s l a t i o n . They a l s o p l a y a
r o l e i n determining t h e r a t e of m y o f i b r i l l a r p r o t e i n s y n t h e s i s i n d i r e c t l y .
T. R. Dutson: I n
mechanism f o r turnover
c e r t a i n length of time
t h e i r a c t i v i t y so they
W . R . Dayton:
t h i s l i g h t , do you t h i n k t h a t maybe t h i s i n t e r n a l
i s necessary i n t h a t a f t e r a c e r t a i n age o r a
do yc)u t h i n k mybe t h e s e p r o t e i n s might l o s e
need t o be t u r n e d over.
Oh, I t h i n k t h e r e i s no doubt about t h a t .
T . R . Dutson: So a c t u a l l y t o t u r n off p r o t e i n degradation or t o
t u r n it completely off might be r e t r o a c t i v e and a c t u a l l y have a slowing
e f f e c t on s y n t h e s i s .
W. R . Dayton: I don't want t o imply that we should completely
t u r n o f f p r o t e i n degradation, but i n c e r t a i n cases a n i m l s may have a
r a t e of p r o t e i n degradation that i s elevated over what i s considered
t h e norm f o r t h e population. But t h e s e a r e t h e kinds of t h i n g s that
we need t o f ind out.
W. G Bergen: Well, from a more p r a c t i c a l view-point, t o increase
p r o t e i n s y n t h e s i s i n meat a n i m a l s f i r s t of a l l I ' d l i k e t o say t h a t
from a n u t r i t i o n a l view-point t h e y a r e not going t o improve v e r y much.
The o t h e r a l t e r n a t i v e t h e n i s t o increase t h e amount of p r o t e i n you
can make w i t h your e x i s t i n g apparatus t h a t you have o r you increase
t h e amount of apparatus o r both. The e f f i c i e n c y t h i n g m y be a t h i n g
that you can handle through g e n e t i c s and hormones and, of course, i t ' s
t h e kind of t h i n g t h a t DES is involved w i t h i n , a l s o s t e r o i d s and
other t h i n g s . So, I t h i n k it i s p r e t t y d i f f i c u l t t o cause a quick
increase i n p r o t e i n deposition, p a r t i c u a r l y i n terms of n u t r i t i o n .
T. R e Dutson: Ron, would y3u l i k e t o speak t o t h a t i n terms of
myogenesis and some of t h e work that you are doing?
R . E . Allen: I would guess t h a t maybe t h e quickest way t o enhance
p r o t e i n s y n t h e s i s would be t o increase s a t e l l i t e c e l l p r o l i f e r a t i o n , i f
y ~ could
u
increase s a t e l l i t e c e l l p r o l i f e r a t i o n and f u s i o n , you would
a u t o m a t i c a l l y have a n increase i n p r o t e i n s y n t h e s i s . I t h i n k you would
have a b e t t e r chance a t m a n i p u h t i n g t h e p r o l i f e r a t i o n of c e l l s t h a n
you would manipulating t h e p r o t e i n s y n t h e t i c a c t i v i t y by a l t e r i n g ribosomes i n i t i a t i o n f a c t o r s , e t c
w i t h i n the cells.
.,
In other words, you could be adding more s y n t h e t i c
T . R . Dutson:
machinery i n t 9 t h e muscle c e l l by f u s i o n with t h e s a t e l l i t e c e l l s .
R . A . F i e l d : I ' d l i k e t o a s k Bob F i t t s a question, I assume t h e
reasgn you were i n t e r e s t e d i n working with miniature pigs was t h a t you
thought perhaps t h e r e would be a species d i f f e r e n c e between them and
t h e rats you've been working w i t h . V i t h regard t o e x e r c i s e , can you
s e e any s p e c i e s d i f f e r e n c e s with regard t o c e l l type changes o r anyt h i n g e l s e you would l i k e t o comment on?
R . H. F i t t s : The reason I s t a r t e d working with miniature pigs
was that most of t h e e a r l y work had been done on small rodents; very
l i t t l e on humans or large; animals; I wanted t o s e e i f t h e response
t h a t i s found i n m d e n t s could a l s o be produced i n l a r g e r animals. I
t h i n k , a t t h i s p o i n t , w e can s a y that t r a i n i n g i s s i m i l a r t o rodents
not only i n miniature pigs but humans a l s o .
R. A . J?ield: Well, that was my question, i f t h e p a r t i c u l a r muscle
has an intermediate f i b e r t y p e i n one animal, and a white f i b e r type i n
another, a r e t h e y both going t o t u r n toward more oxidative c a p a c i t y with
exercise?
3. H. F i t t s : I t h i n k that i s t r u e f o r 211 f i b e r types t h a t a r e
included. I s a y that because i f you a r e running on a f l a t t e r r a i n f a s t
t w i t c h white f i b e r s a r e not included very o f t e n . So, t h e changes i n
t h a t f i b e r types w i t h l e v e l running may be minimal; but with slope
running or h i l l running or any s p r i n t t y p e running then a l l f i b e r types
a r e increased i n ox i d a t ive capacity
.
R . A. Field:
Is t h e r e a n age d i f f e r e n c e as t o where t h i s occurs?
R . H . F i t t s : A s f a r as age is concerned, it is just darn d i f f i c u l t
t o t e a c h an old animal how t o run; i t ' s very hard t o t r a i n such animals.
But, what we have found with longevity s t u d i e s i s t h a t e x e r c i s e s r e t a r d s
some of t h e age changes. With age, your a b i l i t y t o consume oxygen
decreases; with t r a i n i n g you r e t a r d t h e decrease, your a b i l i t y t o
consume oxygen i s enhanced. I n a sense you allow yourself t o do more
work and allow yourself t o filnction b e t t e r i n your d a i l y r o u t i n e as you
get older.
T . R . Dutson: I'd l i k e t o ask another question of Ron Allen i n
terms of t h e involvement of s a t e l l i t e c e l l s and t h e increase i n p r o t e i n
production by t h a t method. Do you have any ideas as t o what might
increase t h e amount of s a t e l l i t e c e l l f u s i o n and would t h i s maybe b e
r e l a t e d back t o some of t h e environmental f a c t o r s t h a t you t a l k e d about
i n myoblast f u s i o n , s m h as collagen, and t h i s s o r t of t h i n g . k y b e we
could increase some of t h e s e e n v i r o n m n t a l f a c t o r s and g e t t h e s e s a t e l l i t e
c e l l s t o f u s e more?
273
R . E . Allen:
I t h i n k t h a t of t h e environmental f a c t o r s I mentioned,
I d o n ' t t h i n k anybody has any d a t a on t h a t . a u t t h e r e was some specul a t i o n on my p a r t a t one time that DES m i g h t possibly increase t h e
p r d i f e r a t i o n of myDgenic c e l l s and ire played around with t h i s off and
on f o r a while. The r e s u l t s a r e very confusing i n that sometimes you
do g e t enhancement of c e l l p r o l i f e r a t i o n assuming t h a t t h e embryonic
myogenic c e l l s a r e good models for s a t e l l i t e c e l l a c t i v i t y . But then
you can a b o l i s h t h i s by s h i f t i n g t h e serum, e t c . , S D t h e experiments
a r e somewhat inconclusive.
T . R . Dutson: Do w e have any more comments? It looks l i k e o w
time i s running a u t , so, I ' d l i k e t o thank t h e gentlemen f o r a f i n e
presentation and a very i n t e r e s t i n g discussion.
* * *
J. D. K e q : The purpose of an update s e s s i o n i s t o b r i n g t o t h e
group t o p i c s of c u r r e n t i n t e r e s t . There is a l o t of i n t e r e s t i n
microbiological standards and t h e reduction of microbiol population i n
f r e s h meats. Also t h e s e populations a r e a f f e c t e d by t h e handling of
carcasses i n shipping. Therefore, our f i r s t speaker w i l l d e a l w i t h
c h l o r i n e washing of carcasses while our second speaker w i l l d e a l with
a l g i n a t e coatings f o r carcasses. We w i l l round out t h e s e s s i o n by
dealing with a t h i r d t o p i c , Forage-Finished Beef. This has been on
t h e minds of m n y people s i n c e t h e p r i c e of beef has tumbled while t h e
p r i c e of g r a i n and supplement has increased.
Our f i r s t speaker is Dr. A . W. (Tony) Kotula. Tony i s Chief, Meat
Research Laboratories, ARS, USDA, B e l t s v i l l e
Tony's t o p i c is "Chlorir,e
Washing of Carcasses .'I