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NUTRITIONAL REGULATION OF MACROM0I;ECULAR SYNTHESIS I N MUSCLE*

IBKNER G . BERGEIi Michigan S ta t e University

INTRODUCTION

Detailed reviews on t h e ove ra l l mechanisms of protein synthesis ( t r ans l a t ion ) have recent ly 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). 1974) and Bergen (1974) have published reviews on t h e regulat ion of protein synthesis and growth i n mammalian muscle. w i l l place emphasis on some of t he mater ia l covered above and w i l l a l so focus on some of the more recent research (and new problems) i n protein synthesis regulation, espec ia l ly as it appl ies t o muscle.

Young (1970,

The present review

The following topics 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 ac to r s and mRNA binding, elongation and coordinate synthesis of the various muscle pro te ins .

Localization of ribosomes i n the m y D f i b e r and t h e i r po ten t i a l r o l e i n t h e spec i f i c i ty f o r myofibr i l lar and soluble muscle protein synthesis.

( 3 )

(4 ) Muscle protein turnover.

( 5 ) The r o l e of developmental and other hormones on muscle protein synthesis .

(6 ) Overall developmental aspects, ce l lu l a r i t y , nucleic acid metabolism and eff ic iency of protein synthesis during muscle growth.

( 7 ) Problems i n studying t h e r o l e of nu t r i t i on and hormones i n muscle protein synthesis mechanisms.

* Presented a t the S t h Annual Reciprocal Meat Conference o f t he American Meat Science Association, 1975.

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Regulation of protein synthesis i n muscle (eukaryotic) c e l l s

Extensive work of the mechanism of protein synthesis has been conducted w i t h bac t e r i a l (prokaryotic ) systems. be divided in to two areas: t ranscr ipt ion, i . e . , the DNA directed generation of genetic messages (mRNA) and the synthesis of the machinery f o r protein synthesis (rRNA and tRNA); and t rans la t ion , i . e . , the decoding of the message in to proteins .

Protein synthesis can

Translation can be divided in to 3 separate steps: I n i t i a t i o n , elongation and termination. binding t o the small ribosomal subunit and the 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 the large r i b o - somes subunit. This process i s then followed by elongation, the step- wise addition of a s ingle amino acid (v ia AA-tRNA) i n t o the s t a r t ed peptide chain as the message i s decoded. Finally, upon completion (or upon reaching a termination codon), the macromolecule is released. This process i s cal led termination.

I n i t i a t i o n involves the mechanisms of mRNA

Haselkorn and Rothman-Denes (1973) concluded that t rans la t iona l mechanisms of protein synthesis a re r e l a t ive ly s i m i l a r between prokaryotic and eukaryotic c e l l s . Based on the 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 overa l l mechanisms of ske l e t a l muscle protein synthesis a r e s imilar t o the process extensively described f o r bac te r i a l or l i v e r systems.

A number of fac tors that regulate or might be r a t e l imit ing 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 ava i l ab 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 fac tors and other fac tors and enzymes

( 6 ) Hormones and hormonal l i k e fac tors

A major long term fac tor i n the 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). t o the substrate supply and there i s a high correlation between t i s sue RNA l e v e l and r a t e of protein synthesis (Wannemacher, 1972; Allison et e., 1963; Wannemacher e t al., 1971). supply i s r e s t r i c t ed t o a growing rat , muscle RNA declines and protein synthesis declines (Howarth, 1972). protein synthesis in l i v e r and muscle of fed o r fasted rats. workers concluded that a decrease i n protein synthesis t o fas t ing was

The amount of t i s s u e rRNA is d i r e c t l y re la ted

When the d ie ta ry amino acid

Henshaw e t al . , (1971) studied These

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due t o a decrease i n ce l lu 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. (1970, 1972) and Omstedt and Von der Decken (1972) studied the e f f ec t of protein s tarvat ion and n u t r i t i v e qua l i ty (Mpu) of d ie ta ry proteins on prDtein synthesis -- i n v i t r o i n r a t ske l e t a l muscle. t h a t when an inadequate protein d i e t ( e i t h e r l e v e l o r nu t r i t i ve qua l i ty ) was fed t o r z t s , there was a decrease i n ribosomal RNA i n the muscle t i s s u e as wel l a s i n t h e a c t i v i t y of t he ribosomes.

Von der Decken and Omstedt

It w a s demonstrated

Some other n u t r i t i o n a l f ac to r s have been re la ted t o protein synthesis i n muscle c e l l s . enhanced by oxidizable non carbohydrate subs t ra tes (Rannels e t a l . , 1974). of spec i f ic muscle proteins ( S h a r d and Srivastava, 1974).

Protein synthesis i n perfused rat hear t muscle was

A vitamin E deficier'cy i n r abb i t s caused s h i f t s i n t he synthesis

The amount of avai lable mRNA can a l so control ove ra l l protein synthesis; t h i s has been especial ly wel l demonstrated i n b a c t e r i a l systems which have mRNA w i t h short h a l f - l i f e (Haselkorn and Rothman- Denes, 1973). demonstrated by an aggregation of ribosomes on mRNA which r e su l t s i n polysome formation (Munro, 1970). ribosomes in to polysames may be interpreted t o be due t o synthesis of new mRNA species or a n enhanced recruitment of already ex is t ing mRNA and possibly other f ac to r s . I n extensive work with the chick oviduct system and the e f f e c t of various hormones ( e .g . estrogen, progesterone and tes tos te rone) on the increase i n the synthesis r a t e of spec i f ic proteins , it has been shown that there may be a n increase i n IuREA synthesis . merase 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 Haines, 1973).

When protein synthesis commences t h i s can usual ly be

Unfortunately the aggregation of

There a l so may be an increase i n DNA dependent RNA poly-

Myosin mRNA has been i so la ted from chick embryo muscle and p a r t i a l l y characterized (Morris e t g. , 1973). (Heywood and Rich, 19687 showed that t h e proportion of the 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 closely resembles t h e pa t te rn of t he appearance of th ick filaments and myosin accret ion i n the developing c e l l (Hermnn 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 & e., 1969). protein synthesis i n muscle was i n pa r t ascribed t o changes i n RNA polymerase a c t i v i t y (F lo r in i and Breuer, 1966). The above r e s u l t s indicate t h a t mRNA a v a i l a b i l i t y plays a major regulatory ro l e i n muscle protein synthesis.

T h i s group of workers previously

Further t h e enhaacing e f f e c t of growth hormone on

Young (1974) has reviewed the l i t e r a t u r e on the ro le of aminoacyl- t R N A on protein synthesis i n muscle. muscle aminoacyl-tRNA synthetase a c t i v i t y did not decrease (Young, 1974); hmever, the a c t i v i t y of t h i s enzyme decreased i n t he l i ve r (Stephen, 1968). nucleotide and sequence (-pcpcp~) on amino acid accepting capacity of tRNA (Deutscher, 1973). published f o r muscle ce l l s , there i s the d i s t i n c t poss ib i l i t y t h a t the r a t e of protein synthesis i n muscle c e l l s may be modulated by the ava i l ab i l i t y of tRNA which can accept amino acids (Young, 1974). The role of soluble ( in i t i a t ion , elongation, termination and interference) fac tors and developmental and other hormones w i l l be discussed i n the topic sections below.

Upon dietary protein depletion,

Others have speculated on the ro le cJf the 3 ' - t r i -

Although def in i t ive r e su l t s have not been

In i t i a t ion , spec i f i c i ty of i n i t i a t i o n fac tors and mRNA binding, elongation and coordinate synthesis of various muscle proteins .

Three s e t s of protein fac tors are involved i n t he process of protein synthesis and i n the proper decoding of the genetic message. When isolated ribosomes a re washed with NH4Cl o r KC1, th ree protein fac tors are removed (Lucas-Lenard and Lipman, 1971). The absence of these factors , t rans la t ion of n a t u r a l mRNA i n the presence of ribosomes, GTP, tRNA, amino acids, ATP e t c ., i s a l so blocked.

Since the i n i t i a t i o n process must involve a discriminatory mechanism t o control which proteins a re t o be synthesized, (e.g. t rans la t iona l control) extensive work has been conducted on the ro l e of i n i t i a t i o n fac tors on the overa l l r a t e and products i n c e l l f ree , natural messenger, protein synthesis systems. In t e re s t i n elongation fac tors i n muscle work has centered on the r o l e of substrate supply on the elongation cycle and protein synthesis r a t e .

Although it had been generally accepted t h a t the regulation of protein synthesis occurs primarily a t the l e v e l of t ranscr ipt ion, recent evidence i n prokaryotic (as well as eukaryotic) systems has suggested t h a t protein synthesis may a l s o be controlled during the t rans la t ion stage-especially i n i t i a t i o n . factors , e.g., IF1, IF2, IF3 (Lucas-Lenard and Lipman, 1971). i n i t i a t i o n , IF3 promotes the binding of natural mRNA t o the small ribo- soml subunit (30s or 40s) (Wefssbach and Brot, 1974; Vermeer - e t a 1 1973). two fo ld by IF2. mRNA complex. the small subunit t o prevent large subunit (50 or 60s) from binding (Weissbach and Brot, 1974). The ro le of the ribosome and IF3 i n the binding of the i n i t i a t o r codon (AUG) i s presently a very ac t ive area of research. ( o r Met-tRNAf i n eukaryotes) i n t he presence of GTP t o the IF3 small subunit mRNA complex. i n i t i a t o r Met-tRNAf and w i l l discriminate against the Met-tRNAm used

There a re three i n i t i a t i o n During

This binding of mRNA t o the small subunit i s stimulated about

IF3 also appears t o promote a conformational change i n This act ion forms a s table ribosomal IF3-30s ( o r 40s)

IF1 and IF2 then f a c i l i t a t e t he binding of t he f-Met-tRNAf

IF2 is highly specif ic f o r binding of the

251

fm- in t e rva l methionine decoding. complex, IF3 i s released, then f i n a l l y t h e GTP is hydrolyzed, the la rge subunit j o i n s the small subunit and IF1 and IF2 a r e released (Weissbach and Brot, 1974).

A s the Met- tRNAf i s bound t o the

I n prokaryotic systems, IF3 appears t o recognize d i f f e ren t c i s t ron i n i t i a t i o n s i t e s on polycis t ronic messages (Revel -- e t a1 ., 1970; Berrassi e t g. , 1971). (i f a c t o r ) has been i so la ted (Groner e t a1 ., 1972). depresses the r eac t iv i ty of IF3 w i t h ce r ta in na tura l W A .

In addition, a protein, cal led the interference f ac to r The i fac to r

Heterologous and homologous t r ans l a t iona l systems (embryonic muscle, re t icu locytes ) have been used t o study t h e spec i f i c i ty of mRNA binding. Tihen r e l a t i v e l y small amounts o f myosin mRNA or 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 ac to r s were required from the same c e l l type a s the messenger t o insure decoding (Thompson and Heywood, 1974; Thompson e t -- a l . , 1973). subsequently shown t h a t IF3 was responsible i n some manner i n mRNA recognition (Heywood e t al . , 1974). More recent ly a low molecular w e i g h t RNA has been i so la ted from i n i t i a t i o n f ac to r (ribosomal sa l t washes) which appears t o be involved i n t r ans l a t iona l regulation of pro te in synthesis (Heywood e t a l . , 1974; Bogdanousky -- e t al . , 1973). This RNA ca l led t r ans l a t iona l cont ro l RNA (tcRNA), i s not e f f ec t ive i n blocking mRNA binding t o ribosomes ( i n i t i a t i o n ) i n homologous systems, but muscle tcRNA causes an abortive i n i t i a t i o n of globin synthesis i n re t icu locyte lysate systems (Kennedy -- e t al . , 1974).

It was

The r o l e of elongation f ac to r s i n muscle protein synthesis has been primarily studied during protein depletion. have concluded that elongation fac tors (EF) l i m i t protein synthesis i n muscle c e l l f r e e -- i n v i t r o systems from protein depleted r a t s . l imi t ing r o l e in vivo of EF i n muscle protein synthesis has not been establ ished (Young, 1974) espec ia l ly s ince a l l c e l l f r e e muscle protein synthesis systems do not exhibi 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 out . Further, the high ionic s t rength buf fers 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 ac to r s ( IF) from the polysome, and thus may ob l i t e r a t e po ten t i a l differences i n i n i t i a t i o n rates between muscle c e l l s from normal and protein depleted animals. i n i t i a t i o n and maybe I F should be r a t e l imi t ing during periods of protein deplet ion.

Young and coworkers (1974)

A r a t e

It would seem most l og ica l t h a t

Much emphasis has been placed on research on the r e l a t i v e synthesis and degradation r a t e of l ight and heavy chains of myosin. In 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 rans la ted on d i f f e ren t mRNA's and subsequently assembled t o form nat ive myosin (Sarkar and Cooke, 1970; Low - e t ,*, a 1 1971; Brivo and Flor in i , 1971). I n adul t s k e l e t a l muscle there 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 turn- over r a t e of heavy myosin chain w a s about twice that of t h e light myosin chain, however, turnover of t o t a l myosin protein re f lec ted t h e

heavy chain myosin a s it accounts f o r a t l eas t 80$ of the t o t a l protein (Wikman-Coffelt e t a l . , 1.973).

Localization of ribosomes i n the myofiber and t h e i r poter, t ial ro le i n the spec i f ic i ty fo r myofibrillar and soluble protein synthesis

I n ty-pical ce l l s , the bulk of protein synthesis occurs on micro- somal ribosomes. Nuclei and mitochondria are among other ce l lu la r components tha t exhibit pmte in synthesis. Flor ini (1964) reported tha t the r a t e of protein synthesis of muscle microscomes was much lower than tha t of l i v e r microsomes; however, muscle mitochondria were a s act ive as l i v e r microsomes (McLean & Q., 1958). the bulk of muscle RNA sediments in the myofibril and nuclei fraction, unlike l i ve r where the highest levels of RNA are found i n the microsomes, (Narayanana and Eapen, 1973; Hulsman, 1961). The extent of RNA recovery i n the various fract ions of muscle i s dependent upon the ionic strength of the homogenizing buffer. Heywood -- e t a l . (1968) showed tha t the ribosome yield from muscle could be vast ly improved by using high ionic strength buffer, as muscle polysomes coprecipitated w i t h myosin during homogenization of the t i s sue i n lower ionic strength buffers. Zak e t -- a l . (1967) isolated the RNA associated with myofibrils of chick embryonic hear t . firmly bound t o the myofibrillar structure and 85% of the RNA was i n the form of ribosomal RNA and s h i l a r t o microsomal rRNA. Recent electron-micrograph work has ten ta t ive ly indicated, that these ribosomes a re associated with the myosin filaments (Larson e t a l . , 1969). 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 par t by microsomes o r by a t o t a l l y independent system. isolated myofibrillar ribosomes are act ive i n protein synthesis and a r e not dependent on c e l l sap factors fo r t he i r ac t iv i ty . demonstrated t h a t myosin is assembled by myofibrillar rRNA and concluded t h a t there i s extensive and independent protein synthesis by myofibrils, with the myofibrillar ribosomes as the functional un i t s . microsomal preparation from muscle (without high ionic strength buffers ) presumably produce nonmyofibrillar proteins. synthesis i n such c e l l f ree systems is usually small ( R . Bjerke, 1974, Iowa State University--personal communication ) .

On fractionation,

These workers concluded t h a t most of the RNA was

Winnick

Narayanan and Eapen (1973a,b) have shown tha t

They also

The usual

The amount of rqyosin

In i t i a t ion factor-3 (IF-3) has been implicated as an obligatory component of mRNA binding t o the small ribosomal subunit (Vermeer e t a l . , 1973; Weissbach and Brot, 1974) and it has been proposed tha t IF3 plays a ro l e i n messenger selection and spec i f ic i ty of protein synthesis. Thus, Heywood & &. (1974) and Thompson and Heywood (1974) have proposed that IF3 governs a f ine tuning mechanism for the synthesis of myofibril lar and sarcoplasmic proteins i n musCle ce l l s . On the other hand, the f a c t t ha t ribosomes a r e located i n specif ic s i t e s i n muscle c e l l s w o u l d argue tha t the ce l lu la r location of ribosomes may a t l ea s t play a role i n the spec i f ic i ty of protein synthesis. More recent work

253

i n r a t l i v e r has shown t h a t d i f fe ren t populations of ribosomes a re segregated i n the c e l l ra ther than a s widely held tha t extensive exchange takes place among ribosomes within a c e l l (Arora and Robert, 1974). This ce l lu l a r s i t e suggestion has some support from the finding (Srivastava and Chaudhary, 1969) that the ribosomes associated with the nuclei-myofibril f rac t ion (primarily involved i n s t ruc tu ra l protein synthesis) declined 5 fo ld from b i r t h t o old age, while t he rRNA associated with the supernatant, microsomes and mitochondria only declined 2-2.5 fold over t h e same period. I n Later l i f e (post growth), it would appear t h a t t he synthesis of sarcoplasmic and soluble proteins (enzymes involved i n energy metabolism, e t c . ) would be r e l a t ive ly more important than the synthesis of s t ruc tu ra l proteins.

Muscle protein turnover

The concept of a dynamic state of t i s sue and the cycling and t u r n - over of t i s sue components w a s firmJ.y introduced by Schoenheimer (1942). The process of protein degradation i s quant i ta t ively almost as important as protein synthesis (Swick and Song, 1974).

A k ine t ic analysis of the processes of protein synthesis and degradation has been made by Schinke (1970). i s a zero-order process, e.g. not dependent on t h e concentration of reactants, while the degradation r a t e i s a first order process, e.g. dependent on the concentration o f the reactants . Work i n protein degradation has a l so shown t h a t the d i f fe ren t proteins have t h e i r own spec i f ic r a t e constant which appears t o be a property of the specif ic protein and i ts interact ion with other ce l lu l a r fac tors (Swick and

He showed that synthesis

Song, 1974).

A major problem f o r a l l s tudies on in vivo synthesis o r degradation rates of proteins with radioisotopes, especially a s ingle dose admini- s t ra t ion , is a proper analysis of the spec i f ic a c t i v i t y of the precursor pool, and a l so the recycling and r eu t i l i za t ion of labelled amino acids . To overcome the l abe l r eu t i l i za t ion problem, a var ie ty of approaches have been developed t o insure removal of t he label led amino acid from t h e precursor pool a f t e r one cycle. Two common approaches t o overcome re labe l l ing problems have been t o flood the system with the unlabelled compound and/or feeding high protein d i e t s or t such as guanidino 14C labelled arginine o r Na$CO3 as alanine and glutamic acid, which have a high metabolic degradation r a t e and, thus, a re l e s s extensively reu t i l i zed (Swick and Song, 1974; Schimke, 1970). approach of overloading with unlabelled amino acids (chase) appears t o be somewhat undesirable as the feeding of high protein d i e t s o r s ing le amino acids may induce a physiological response t h a t w i l l i n t e r f e re with the desired r e s u l t s .

use a protein precursor

The

2 54

Most ear ly work on protein degradation was done i n l i v e r and some of the procedures used i n l i v e r a r e not applicable i n muscle. Thus, w i t h s ingle inject ion experiments it was shown tha t labelled arginine was extensively reu t i l i zed (Millward, 197Ck; Swick and Handa, 1956). Labelled (l i4-C) glutamic acid appeared t o be a n excellent compound t o study degradation i n muscle protein and gave r e su l t s similar t o l 4 C carbonate inject ions (Swick and Song, 1974). protein def ic ien t d e t or a r e i n a wasting s t a t e , the use of a s ingle administration of l'&-glutamate w i l l not give val id estimates of protein turnover since the l abe l was extensively recycled. Under these circum- stances, cumbersome techniques of infusion or perfusion become necessary where the spec i f ic acid or the labelled species i n question i n the soluble pool can be continuously monitored (Swick and Song, 1974).

When animals a re fed a

The ef fec t of s tarvat ion, law protein d i e t s , hormones and induced muscle hypertrophy on ske le t a l mixed muscle protein synthesis and degradation has been studied. Starvation or feeding protein f r ee d i e t s decreased the r a t e of synthesis and increased t h e r a t e of breakdown of proteins i n muscle of r a t s (Millward, 1970a; Young e t a1 ., 1971). When r a t s were refed an adequate protein d i e t , i n i t i a l l y the degradation of muscle proteins was stopped and synthesis of proteins was increased, but a f t e r a long period of refeeding, the mixed ske le t a l muscle protein degradation r a t e returned t o normal (Young e t al . , 1971).

I n injury induced hypertrophy of rat soleus muscle, there was decreased catabolism and increased synthesis of mixed muscle proteins (Gcldberg, 1969a ) . Furthermore, the degradation of t he soluble ( sarco - plasmic) proteins decreased more markedly than that of the myofibril lar proteins resu l t ing i n a r e l a t ive increase of sarcoplasmic protein accretion during injury induced compensatory growth (Goldberg, 1969a). Growth hormone, however, increased protein synthesis i n l eg ske le t a l muscle without changing the protein degradation r a t e (Goldberg, l g 9 a ) . When rats were t rea ted w i t h cortisone, there w a s an increase i n degradation of mixed proteins of ske le t a l muscle (Goldberg, l969b).

During hypertrophy induced by muscle denervation of r a t diaphrams, the r a t e of protein synthesis i n i t i a l l y increased t o more than twice the normal r a t e and then declined t o 5G$ of the normal r a t e , however, the r a t e of mixed muscle protein degradation a l so increased t o more than twice the normal r a t e of ske le t a l muscle (Turner and Garlick, 1974).

The turnover r a t e constant as well as h a l f - l i f e of proteins i s (as outlined above) dependent on the extent of labe l recycling. recycling occurs during a study, the lower the degradation r a t e and the longer the half - l i f e . reported t h a t the average half - l i f e of mixed ,muscle protein w a s 5-6 days. Half-lives fo r muscle sarcoplasmic and myofibril lar proteins i n normal r a t s were 3.6 and 15.6 days, respectively (Millward, 1970b). pig ske le t a l muscle, the ha l f - l ives for mixed sarcoplasmic and mixed myofibril lar proteins were 9.4 and 16.4 days, respectively (Perry, 1974).

The more

W8terlow and Stephen (1968) and Garlick (1969)

I n

255

The h a l f - l i f e for intramuscular connective t i s sue proteins i n pigs was 20 days (Perry, 1974). purified myosin and ac t in have been shown t o be i n the range of between 20-45 and 50-70 days, respectively (Swick and Song, 1974).

The half- l ives f o r rat ske le t a l muscle f o r

The ro le of developmental and other hormones on muscle protein synthesis

The hormones which play a ro le i n the development and growth of muscle a re growth hormone ( G H ) , insul in , androgens and thyroxine. Although these hormones promote protein synthesis mechanism, they a re not an obligatory component of the protein synthesis mechanism. accretion during work induced hypertrophy has been found i n hypophy- sectomized rats (Goldberg, 1967) and Leathem and Koishi (1972) reported tha t GH or insul in were not needed f m the replet ion of t i s sue proteins .

Protein

The mecbnism(s) by which hormones stimulate protein synthesis i s not well detai led. The operon, regulator gene, repressor and derepressor hypothesis of Jacob and Monod (1961) which predicted the existence of an unstable mRNA as a mechanism fo r qua l i ta t ive and quant i ta t ive control of protein synthesis w a s applied t o hormonal regulation. proposed t h a t hormones might a c t as a derepressor. of higher animals the regulation of protein synthesis occurs a t many s i t e s other than mRNA synthesis (Williams-Ashman and Reddi, 1972). ( 1968 ) has proposed tha t developmental hormones (and androgens ) promote t i s sue protein synthesis by enhancing the synthesis of ribosomes (protein synthesis machinery). terone increased the template a c t i v i t y of DNA i n chromatin and a l so protein synthesis i n ra t ske le t a l muscle. Androgens did not, however, promote the synthesis of any spec i f ic muscle proteins (Flor ini , 1970).

Thus, it was However, i n ce l l s

Tata

Breuer and Flor in i (1965,1966) found t h a t tes tos-

Insul in promotes protein synthesis by a mechanism independent of i t s ro le i n glucose metabolism (Manchester, 1972). The insu l in e f f ec t i n muscle i s e i ther re la ted t o amino acid uptake (Goldstein and Reddy, 1970), but discounted by London (1973) and Manchester (1972), or t o a d i r e c t e f f ec t on the t rans la t ion (Manchester, 1972). The ro l e of insul in i n muscle protein synthesis is not limited t o DNA dependent RNA synthesis (Woll, 1972) i n contrast t o androgens and developmental hormones. Growth hormone appears t o primarily influence t i s sue protein 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 ro l e of thyroxine i n muscle protein i s not c lear ; however,

decreased the r a t e of elongation i n ra t l i v e r (Ma'chews _. ,*,

Overall developmental aspects, ce l lu la r i ty , nucleic acid metabolism and efficiency of protein synthesis during muscle growth

The overa l l growth pat tern of ske l e t a l muscle (Enesco and Puddy, 1%; Chiakulus and Pauly, 1965) has been outlined from studies with rats and mice. During ea r ly postnatal growth (pe r ina t a l ) there i s a

3 f o l d increase i n t o t a l DNA and nuclei i n muscle, whereas muscle mass increases approximately 4-5 fo ld . This period is generally considered one of hyperplasia and some hypertrophy. there i s only a f i f t y percent increase i n nuclei and DNA content while muscle mass increases about 3 fo ld . This period is generally cal led hypertrophy. number of myofibers per muscle, hmever, remain constant. The increase i n DNA i n muscle during growth has been a t t r ibu ted t o mitosis i n undifferentiated myoblasts, s a t e l l i t e c e l l s and other c e l l s (Moss and Leblond, 1971; Mauro, 1961), while it is generally accepted t h a t myofiber numbers a r e established p r io r t o b i r t h or during the per ina ta l period and growth i n muscle mass occurs through increases i n individual myofibers (Young, 1970; Goldspink, 1972).

I n the young adult stage

Throughout this period ( b i r t h t o young adul t s tage) t he

Changes i n muscle nucleic acid and protein content o r concentrations have also been studied w i t h a number of meat producing animals. pigs (Robinson, 1969) and sheep (Johns and Bergen, 1973) t o t a l DNA, RNA and protein increase during muscle growth. however, decline continuously from b b t h t o a f i n a l l e v e l due t o the rapid increzses of other ce l lu l a r components (especial ly protein) of muscle. I n normal growth, i n pigs and sheep, changes i n RNA and DNA were para l le l , while the protein/DNA or protein/RNA r a t i o s increased markedly (Robinson, 1969; Johns and Bergen, 1973). t o be l i t t l e change i n protein/DNA r a t i o (an indication o f c e l l s i z e ) during l a t e r growth s tages . 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" ca t t l e , muscle protein/DNA and RNA/DNA r a t io 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 lean s t r a i n of pigs had consistantly higher RNA/DNA and protein/ DNA r a t i o s than a fa t s t r a i n of pigs (Poland China). Powel and Aberle (1975) showed, however, t h a t overall , i n heavy and l i gh t muscled Duroc pigs, protein/DNA and RNA/DNA r a t i o s differed l i t t l e . during normal growth (adolescence), the DNA and RNA concentrations i n muscle decline markedly and then s t a b i l i z e i n the adult , while the protein/HNA reaches i t s plateau value. It appears t h a t increases i n muscle mass are DNA (e.g. nuclei 7 . An induced pro l i fe ra t ion of ruuscle nuclei during postnatal 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.

I n both

Nucleic acid concentrations,

There a l so appears

I n summary then,

eneral ly accwpanied by a proportional increase i n

This general pattern of constant c e l l s i ze (protein/DNA r a t i o ) Thus, when sheep can be modified by nu t r i t i ona l s t r e s s in animals,

were fed a low (7%) protein ra t ion during a 60 day, post weaning, feeding t r i a l , muscle RNA/DNA r a t i o s declined but protein/DNA r a t i o s were ac tua l ly somewhat increased (Johns and Bergen, 1973). While the lack of substrate (amino ac ids) f o r protein synthesis tends t o depress the production of r R N A (WaMemaCher, 1972) concomitantly the protein degradation is depressed (Millward e t a1 ., 1974) possibly accounting fo r an elevation i n protein/DNA r a t i o s . When rat pups were assigned i n groups of 3, 8 or 16 per dam or malnourished during ear ly postnatal growth, t he rats having the lowest w e i t gain had t he la rges t protein/ 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 relationship between muscle s ize (protein/DNA ra t io , Cheek -- e t al., 1968) and l ive weight gain. author (Stewart, 1974) concluded t h a t the larger the c e l l s ize i n muscle t i s sue a t a par t icular body weight, the lower the potent ia l for fur ther body growth.

The

Efficiency of protein synthesis can be defined as the p o l e s of amino acids incorporated per mg polysomal RNA ( r R N A ) per uni t time. From a long term regulation view, the level of t i s sue r R N A i s the major factor influencing the r a t e of protein synthesis (IJannemacher, 1972) . Millward -- e t a l . (1973) and Henshaw L- e t a l . (1971) showed a d i r ec t re la - t ionship between protein synthesis and t i s sue polysomal RNA content and a highly s ignif icant (pos i t ive) relationship between growth ra tes i n r a t s and the efficiency of protein synthesis in muscle and l i v e r . mechanism fo r t h i s improved r a t e of amino acid incorporation per uni t polysomal RNA is not clear, presumably it i s mediated by soluble c e l l sap factors , hormonal f a c t w s and other factors .

The

I n past work, the number of ribosomes act ively involved in protein synthesis have been measured with polysomal prof i les , polysomal rRNA/ t o t a l RNA r a t io s or as the r a t i o of subunit RNA t o polysomal RNA. increased protein synthesis w a s usually ascribed t o an increased number of polysomes. All polysomal ribosomes may not be active i n protein synthesis, however (Sussman, 1970). and N a n a (1974) studied polysomal prof i les and 11, 14 and 17 day old embryonic chick leg muscles. there i s a 3 fold ( a t l e a s t ) increase i n muscle protein synthesis r a t e . Throughout the study, 9% of the ribosomes were present i n polysomes and, thus, the amount df polysomal RNA cannot account fo r the enhanced r a t e of protein synthesis (Nwagwu and Nana, 1974). These workers estimated the number of t R N A per ribossmes i n the polysome. a ribosome carrying t w o tRNA (one each a t the P and the A s i t e ) would be act ive. 17 day embryonic chick muscle. a lso not different f w the three age groups. Nana, 1974) therefore concluded t h a t the increased r a t e of protein accretion (frm 11 t o 17 days of age) must be related t o an increase i n the efficiency of protein synthesis by the ribosomes. Bergen (1974) depressed growth (weight gain) i n r a t s by r e s t r i c t ing 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 efficiency related t o overal 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 the two groups of r a t s and the extra w e i g h t gain i n the control animals was mostly f a t (Bergen, unpublished).

Thus,

To c l a r i fy t h i s problem, Nwagwu active ribosomes" i n I t

During t h i s period

It was reasoned tha t

The number of "active" ribosomes were similar fo r 11, 14 and Muscle proteolytic ac t iv i ty was low and

These workers (Mwagwu and

Problems i n studying the ro le of nutr i t ion and hormones i n muscle protein synthesis mechanism

Great e f f o r t has been expanded by many workers on the relationship of hormonal and nut r i t iona l changes with protein synthesis i n the

2 58

muscle (see Bergen, 1974; Trenkle, 1974; and Young, 1974). is c lear t h a t lack of substrate (protein or energy) w i l l depress the l e v e l and a c t i v i t y of protein synthesis machinery, there i s l i t t l e in formt ion about the r a t e l imit ing s tep during such a physiological s ta te(s) . The proposed action of t h e various hormones a re often very speculative, derived from i n v i t r o work and a re probably often not applicable t o the -- i n vivo s i tua t ion . A good example of such a problem is the inverse relat ionship between growth r a t e i n s t ee r s and serum GH leve ls (Grigsby, 1973 ) .

Although it

To study overa l l aspects of muscle protein synthesis ra tes (other than an assessment of net accretion during a feeding period) e i ther 5 v i t r o or & vivo, procedures using radiact ive labelled substrates (amino acids ) have been employed.

Although the -_I_ i n vivo approach should yield generally useful r e su l t s , too often research reports appears t h a t use such naive approaches t h a t the r e su l t s a r e useless . Recently, Hsueh e t a l . (1975) reported t h a t muscle protein synthesis r a t e was similar f o r r a t s fed a poor qual i ty protein or high qual i ty protein d i e t , although there w a s a three fo ld difference i n weight gain. Protein synthesis w a s measured as CPM incorporated per 100 mg protein isolated. a l l too common, but t o t a l l y inappropriate. 7- i n vivo ( i n any given organ), the spec i f ic a c t i v i t y 3f the precursor pool during the time course of the incorporation period must be described. T h i s problem i s even more important i f only a t r ace r dose (no cold c a r r i e r ) i s administered. the confines of the "precursor pool"; however, t h e spec i f ic a c t i v i t y of the labelled amino acid i n the ce l lu l a r tEWA, nascent peptides chains ( I l a n and Singer, 1975) or i n the t o t a l acid soluble t i s s u e ex t rac t (Fern and Garlick, 1974) can be measured. determinations i n acyl-tmA and nascent chains a re qui te involved and not often attempted. The t o t a l t i s s u e pool approach does present a useful estimate that i s generally not too d i f fe ren t from the "real" precursor pool (Khairallah and Mortimore, 1975).

This approach is unfortunately To study protein synthesis

There i s considerable disagreement as t o

Specific a c t i v i t y

The extent of the description of the label l ing pat tern of the precursor pool spec i f ic a c t i v i t y (see the following f o r methods and theore t ica l considerations of specif ic a c t i v i t i e s : Garlick and Millward, 1972; Henshaw I- e t a l . , 1971; Airhart e t al., 1974; Garlick Fern and Galick, 1973, 1974; I l an and Singer, 1975; Venrooij e t al . , 1974) depends on the questions posed by the invest igator . For studies on the e f f ec t of a given treatment or variable on several aspects of protein synthesis (e.g. increase, no change o r decrease) r a t e , it need only be shown t h a t t he product's (e.@;. newly synthesized o r completed muscle proteins) radioactive amino acid content i s not merely a function of spec i f ic a c t i v i t y changes in the t i s sue (precursor) f r e e amino acid pool. protein synthesis, the specif ic a c t i v i t i e s of t he t i s sue (precursor) f r e e amino acid pool must be determined. precaution, the overa l l ro le of given experimental variables on precursor pools (amino acid concentrations ) should be studied.

g. , 1973;

For more quant i ta t ive work and calculations of the eff ic iency of

A t t h e same time, a s a good

The i n vitro, c e l l f r e e protein synthesis systems avoid the precursorpool problems, but a host of new problems are introduced. The rout ine i so l a t ion from muscle of microsomal systems (ribosomes ) and soluble f ac to r s necessary f o r c e l l f r e e protein synthesis i s qui te d i f f i c u l t . Low ionic s t rength buffers only remove a s m a l l f rac t ion of t he RNA i n muscle, increasing the ionic s t rength improves the y ie ld markedly; but nevertheless l e s s than half of t he c e l l u l a r RNA i s extracted. The a c t i v i t y of homologms -- i n v i t r o c e l l free muscle systems i s generally qui te l o w (less than 3$ of t h e -- i n vivo r a t e ) prompting invest igators t o use m l y muscle ribosomes and l i v e r soluble f ac to r s and precharged t R N A . The microsomes removed from muscle a t low ionic s t rength are not t yp ica l a s they make f e w f i b r i l l a r proteins . The polysomes extracted a t higher ion ic s t rength (Heywood -- e t a l . , 1968) most l i k e l y a re devoid of i n i t i a t i o n fac tors (e .g. s a l t washed ribosomes, Lucas-Lenard and Lipmann, 1971) and these systems can only f i n i s h t he already s t a r t e d nascent peptide chains. Thus, an i n v i t r o approach t o assess nu t r i t i ona l o r hormonal modulation of protein synthesis i n muscle i s n o t adequate. Carefully, reconst i tuted muscle c e l l free pro te in synthesis systems are , however, very useful i n t h e study of t h e r o l e of IF, elongation fac tors , tRNA, the ro l e of ribosomal proteins, etc. , in muscle protein synthesis; but these systems seldom r e f l e c t any physiological circumstances which a re of primary i n t e r e s t t o t he meat and animal s c i e n t i s t .

Mechanisms of protein synthesis ( t r ans l a t ion ) 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 fec t ive ly u n t i l new experimental approaches have been evolved. During nu t r i t i ona l and hormonal challenges, i n i t i a t i o n , elongation and possibly termination may become rate l imi t ing (cont ro l po in t ) t o t r ans l a t ion . There a re now no ef fec t ive procedures t o study these processes without destroying the physiological and u l t r a s t r u c t u r a l milieu of t he muscle system. Some of t h e newer approaches i n studying i n i t i a t i o n react ions (e .g . N terminal vs . i n t e rna l methionine uptake; Oleinick, 1975), a d i r e c t assay of I F concentrations i n crude muscle ribosome ext rac ts (Lubsen and Davis, 1974), or use of spec i f i c i n i t i a t i o n inh ib i tors (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 instead of using w e l l described c e l l f r e e protein synthesis incubation systems, t h e k ine t i c synthesis machinery ( e .g . myosin, ac t in , spec 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 tudies w i l l become a more appropriate t o o l t o study the e f f e c t of n u t r i t i o n a l changes, hormone secret ions and growth r a t e modifications on t r ans l a t ion and protein deposition in muscle.

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T . R . Dutson: I t h i n k f i rs 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 sk each o ther 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 tar t out with in te rd iscuss ion here and t h i s might s t imu la t e d iscuss ion from t h e audience. A t t h e poin t t h a t people i n t h e audience have ques t ions , if you would just r a i s e your hands, we can have a broad d iscuss ion back and f o r t h .

C . E . Allen: Ron, I 'd l i k e t o a sk you, is 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 ther 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 eve 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 eve 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 rea t e r than what you would f i n d phys io logica l ly .

W. G . Bergen: On t h e question of turnover, it has been suggested that energy is required t o keep t h e calcium under con t ro l .

W . R . Dayton: There i s obviously an energy requirement of t h e SR f o r t ak ing up calcium and s o it is poss ib le that t h e r e is 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, genera l ly 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 cases . There a r e r epor t s of some lysosomal enzymes that a r e a c t i v e a t higher pH's bu t I don ' t t h i n k they a r e extensive .

R. H. F i t t s : In c e r t a i n types of exerc ises , 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 but nothing near ly t h e range t h a t you a r e t a lk ing about.

W . R . Dayton: I guess what I would l i k e t o emphasize is that t h e reason that I dwelled on t h e lysDsoiml th ing s o much is that I t h i n k maybe i n t h e past we looked at"1ysosomes because they were the re , 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 they a r e a l i t t l e t o o genera l t o do t h e kinds of t h ings 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 is a r e a l controversy i n that area now and t h e main reason, I th ink , is 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 us ing , some of them a r e not designed properly i n t h e sense that they 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, they 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 ask 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 myof ibr i l la r p ro te ins and t h e r a t e D f p ro t e in turnover of these pro te ins ; 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 rate of C A F a c t i v i t y on these pro te ins 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 tud ie 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 myof ibr i l la r p ro t e ins r e l a t i v e t o each o ther and i n most cases troponin and tropomyosin a r e found t o t u r n over more r ap id ly than a c t i n and myosin. I don ' t mean here t o imply that CAF does a l l of t h e turnover of myof ibr i l la r p ro t e ins , bu t t h e a c t i v i t y of CAF on myof ibr i l la r p ro te ins is cons is ten t with da t a that has been published t o date 3n t h e r e l a t i v e r a t e of turnover of t hese p ro te ins .

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 clear t h a t t h i s work was done by Dennis Olson, another graduate s tudent and not by me; bu t , we have looked a t t h e poss ib le 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 ind on myof ibr i l la r p ro t e ins 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 acce le ra t ing calcium re l ease , of con t ro l l i ng pH dec l ine and th ings like t h i s that 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 th ings that mechanistically would be p r e t t y d i f f i c u l t t o do and this c e r t a i n l y requi res 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 ap id a t 3 7 O C t h a n a t any o ther temperature; hmever , t h e enzyme is f a i r l y uns tab le a t 3 7 O C , it apparently hydrolyzes, i n f a c t , w e have good evidence that it does. that's a problem i n vivo.

However, we don ' t r e a l l y know whether

T . R. Dutson: In t h i s l i g h t then, if you could have a high enough calcium concentration around t h i s enzyme a t a high enough temperature then postmortem degradation of t hese p ro te ins would t ake place very r ap id ly . Is that r i g h t ?

W . R . Dayton: Yes.

T. R. Dutson: If y ~ u would hold t h e temperature high and i f t h e amount of calcium t h e r e w a s h igh enough t o s t imula te t h i s enzyme a c t i v i t y it would go a t a very r ap id 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 then using SDS ge l s as a marker of t h e degradation, he found t h a t , D r . Goll, correc t me if 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 is not c e r t a i n nov as t o whether it is 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 that 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 . Go l l : In terms of postmortem muscle i n s o fa r as we can d i s t i n g u i s h th ings 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.

C . E. Allen: I presume from what has been sa id then t h a t , f o r 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, increase i n calcium or 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 ap id ly , such as some dystrophies where you have problems with t h e SR where they don't t ake up t h e I wondered if t h e a c t i v i t y of CAF is d i f f e r e n t i n these models.

calcium.

W. R . Dayton: There a r e s e v e r a l people i n our lab working on t h i s now, on dystrophic muscle a t t h e present time and they 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 repa ra t ions ) . calcium requirement of C A F i n t h e dystrophic muscle. know w h a t t h e involvement of calcium is, in other words, t h e f a c t tha t CAF a c t i v i t y is increased does not necessa r i ly 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 that t h e SR does appear t o be impaired i n dystrophic muscle would tend 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 equ i r e higher calcium l eve l s than we t h i n k tends t o be f r e e i n muscle c e l l s .

But I don ' t be l ieve there is any change i n t h e We don ' t r e a l l y

T . R . Dutson: I ' d l i k e t o ge t back t o t h e subjec t of exerc ise and t h e d i f f e rences i n lean 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 the re fo re , 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 this when they 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 they have t o use up body s t o r e s of fat?

R. H . F i t t s : There's a good dea l of work done a t t h e University of Chicago studying the e f f ec t of exercise on c e l l numbers and c e l l s i z e . S t a r t i ng t r a in ing ea r ly in l i f e , they found that if y3u exercise ea r ly i n l i f e , it reduces the numbers of f a t c e l l s that w i l l u l t imately reach a ce r t a in s i z e and t h a t even if you terminate exercise, they w i l l remain decreased, where on t h e other hand, if 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 the 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 ren t type of mechanism going on the re . I th ink it has been observed that ce r t a in 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 ab le t o increase myofibril lar s i z e . It has been reported that there 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 he game. TiJe know that there i s no change in muscle t o body weight r a t i o s .

D. E. Goll: The husband D f a technician working in our l a b bicycles a l D t and he claims the fo lk lore on bicycle racing has it t h a t about 4 days before t h e race 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 is a week before the race, you go out and run long runs and deplete your muscles of glycogen s tores when you go on a low carbohydrate d i e t t o fur ther maintain t h i s . t he race you start overloading, eat ing not necessar i ly more t o t a l ca lor ies but more t o t a l carbohydrates. People make t h e mistake of ea t ing mx-e t o t a l ca lo r i e s , l i k e eat ing 5 loaves of bread; they invar- iab ly 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 the muscle glyccgen 3 and 4 fold over normal l eve l s .

Then 3 days before

F . B. Shorland: There seems t o be one thing t h a t wasn't discussed l a t e l y : it is maybe a l i t t l e off the beat in terms of t h i s meat discussion, but I do th ink t h e study of meat is a ra ther wonderful thing because it does bring us back t o t h e question of how these things e f f ec t humans. So t h e t o t a l e f f ec t on hurnans is qui te grea t through t h e study of meat. worked on a t a l l , but does exercise promote longevity? The other thing that wasn't discussed is t h a t w e keep on losing calcium as we ge t older and we go on losing protein, pa r t i cu la r ly i n rnzles, which is ra ther sad. But I th ink it has been indicated here that i f you kept on exercising, you might br ing back a l i t t l e of t he protein, s o it would be worthwhile t o keep everybody moving. W h a t I would l i k e t o know is , there any re la t ionship t h a t anybody here can see, which is the premise of t he question, namely, i f you go on losing calcium is there any connection t o this and t h e reason why you go on losing protein? care t o comment on tha t?

There i s one point, I don't know whether i t ' s been

Anybody

D. E. Goll: Is t h e calcium from t h e bone or t he muscle?

F. B. Shorland: The loss i s probably from t h e bone, t h e skeleton. Actually, I would l i k e t o see if you can ge t anywhere on t h e lcngevity one and its re la t ionship t o exercise .

C . E. Allen: Well, i n that regard, it is wel l documented t h a t overweight individuals have a shorter half l i f e . When you ge 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 losses 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 think the re ' s no doubt i n my mind t h a t exercise t r a in ing increases ones fee l ing of well-being through changes t h a t take place in the hear t and other muscles. Although there a r e no r e a l good epidemiological s tudies over time, and I might add they a r e very d i f f i c u l t t o cont ro l due t o d i e t a ry interact ions, 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 , de f in i t e ly the qua l i t y of l i f e o r t h e fee l ing of well-being is enhanced, but as far as longevity; I ' m not sure you're going t o get de f in i t e answers on t h a t .

C . E . Allen: I might add t o w h a t Bob has sa id , I th ink he made a statement i n h i s presentat ion 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 rats t h a t a r e exercised f o r d i f f e ren t periods of time and it is very fascinat ing t o go down there and, if you d idn ' t know anything about t h e periods of time t h a t these rats were being exercised, t h e one th ing you would note, just observing t h e cages, i s that those rats that are exercising f o r longer periods of time, usual ly when they get back in 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 is 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 they m i g h t be of a lower body weight, and addi t ional ly , they appear t o have l e s s f a t on the body.

T. R . Dutson: I 'd l i k e t o ask a question of B i l l Dayton and Werner Bergen a l s o might l i k e t o coment on this . Getting back t o the protein synthesis-degradation in te rac t ion and the ne t amount of protein synthesis due t o e i the r an increase o r decrease of e i t h e r one of these, would you care t o speculate on some of t h e mechanisms by which th i s protein synthesis could be increased. I th ink we would l i ke t o have some s o r t of hypothesis come out of this 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 synthesis . hhybe we need t o decrease degradation, maybe we need t o increase synthesis . Is this possible, and, if so , how?

W. R . Dayton: I haven't r e a l l y been in to protein synthesis very much; as far as protein 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 se does know what t h e e f f ec t of protein degradation is on t h e growing animal. capacity t o degrade p m t e i n is de f in i t e ly there; but f o r some reason, t h i s capacity is completely shut off or nearly shut off under normal circumstances. I th ink it would be of i n t e re s t t o f ind out exactly w h a t t h e mechanism is that controls t h e r a t e of protein degradation and whether the rate I s high or low. However, as far as put t ing any

I suppose it could be something signi,ficant because t h e

q u a n t i t a t i v e value on w h a t percent of growth or ne t p ro te in synthes is is due t o degradation, I don't t h i n k we can say . Another t h ing we might t h i n k about too , g e t t i n g away from myof ibr i l la r p ro te ins , 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 ro te ins but it happens t o pro te ins 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 myof ibr i l la r p ro te ins but what happens t o t h e 70 some-odd pro te ins i n t h e lysosomes that m i g h t ge t degraded c)r synthesized. Also, 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 synthes is or degradation and these 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 lay a r o l e i n determining t h e r a t e of myof ibr i l la r p ro t e in synthes is i n d i r e c t l y .

T. R. Dutson: In 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 mechanism f o r turnover 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 c e r t a i n length of time do yc)u t h i n k mybe these pro te ins might l o se t h e i r a c t i v i t y s o they need t o be turned over.

W . R . Dayton: Oh, I t h i n k t h e r e is no doubt about that .

T . R . Dutson: 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

So a c t u a l l y t o t u r n off p ro t e in degradation or t o

e f f e c t on synthes is .

W. R . Dayton: I don't want t o imply that we should completely t u r n of f p ro t e in 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 ro te in degradation that is elevated over what i s considered t h e norm f o r t h e population. But these a r e t h e kinds of th ings 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 ro te in synthes is i n meat an imals 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 they a r e not going t o improve very much. The o ther a l t e r n a t i v e then is t o increase t h e amount of p ro t e in 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. that you can handle through genet ics and hormones and, of course, i t ' s t h e kind of t h ing 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 ings . So, I t h i n k it is p r e t t y d i f f i c u l t t o cause a quick increase i n p ro te in deposit ion, p a r t i c u a r l y i n terms of n u t r i t i o n .

The e f f i c i ency th ing m y be a th ing

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 ro te in synthes is 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 , if y ~ u could 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 fus ion , you would au tomat ica l ly have an increase i n p ro te in syn thes i s . I t h i n k you would have a b e t t e r chance a t manipuht ing t h e p r o l i f e r a t i o n of c e l l s than you would manipulating t h e p ro te in syn the t i c a c t i v i t y by a l t e r i n g ribo- somes 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 t h e c e l l s .

T . R . Dutson: machinery i n t 9 t h e

R . A . F ie ld :

In other words, you could be adding more syn the t i c muscle c e l l by fus ion with t h e s a t e l l i t e c e l l s .

I ' d l i k e t o a sk Bob F i t t s a question, I assume t h e reasgn you were in t e re s t ed 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 fe rence between them and t h e rats you've been working wi th . Vi th regard t o exerc ise , can you see any spec ies d i f fe rences with regard t o c e l l type changes or any- th ing 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 see i f t h e response t h a t is found i n mden t s could a l s o be produced i n l a rge r animals. I th ink , a t t h i s po in t , w e can say that t r a i n i n g is s imi 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 type i n one animal, and a white f i b e r type i n another, a r e they both going t o t u r n toward more oxidative capac i ty 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. tw i t ch white f i b e r s a r e not included very of ten . So, t h e changes i n t h a t f i b e r types wi th 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 type running then a l l f i b e r types a r e increased i n ox idat ive capacity .

I say that because i f you a r e running on a f l a t t e r r a i n fas t

R . A . F i e ld : Is t h e r e an age d i f f e rence 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 each 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 tud ie s is t h a t exerc ises r e t a r d s some of t h e age changes. 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. work and allow yourself t o filnction b e t t e r i n your d a i l y rout ine as you ge t o lder .

With age, your a b i l i t y t o consume oxygen

In a sense you allow yourself t o do more

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 ro te in production by t h a t method. increase t h e amount of s a t e l l i t e c e l l fu s ion 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 that you t a lked about i n myoblast fus ion , s m h as collagen, and t h i s s o r t of t h ing . k y b e we could increase some of these env i ronmnta l f a c t o r s and ge t t hese s a t e l l i t e c e l l s t o fuse more?

Do you have any ideas as t o what might

273 R . E . Allen: I th ink t h a t of t h e environmental fac tors I mentioned,

I don ' t th ink anybody has any data on that . au t there was some specu- l a t i o n on my par 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. 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 . you can abol ish t h i s by s h i f t i n g t h e serum, e t c . , SD t h e experiments a r e somewhat inconclusive.

The r e s u l t s a r e very confusing i n that sometimes you

But then

T . R . Dutson: Do w e have any more comments? It looks l i k e o w time is running aut , 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.

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J. D. K e q : The purpose of an update session i s t o br ing t o t h e group topics of current 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. carcasses i n shipping. Therefore, our f i r s t speaker w i l l d e a l with chlor ine 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. dealing with a t h i r d t o p i c , Forage-Finished Beef. t h e minds of mny people s ince t h e p r i c e of beef has tumbled while t h e pr ice of grain and supplement has increased.

Also these populations a r e affected by the handling of

We w i l l round out the session by This has been on

Our f i r s t speaker is Dr. A . W. (Tony) Kotula. Tony is Chief, Meat Research Laboratories, ARS, USDA, B e l t s v i l l e Tony's top ic is "Chlorir,e Washing of Carcasses .'I