LSHTM Research OnlineSketcher, RD; (1976) The metabolism of the branched-chain amino acids in the rat. PhD thesis,London School of Hygiene & Tropical Medicine. DOI: https://doi.org/10.17037/PUBS.04655241Downloaded from: http://researchonline.lshtm.ac.uk/id/eprint/4655241/DOI: https://doi.org/10.17037/PUBS.04655241Usage Guidelines:Please refer to usage guidelines at https://researchonline.lshtm.ac.uk/policies.html or alternativelycontact researchonline@lshtm.ac.uk.Available under license: http://creativecommons.org/licenses/by-nc-nd/2.5/https://researchonline.lshtm.ac.ukR. D. SKE1CHKRABSTRACT OF Ph.D THESISMETABOLISM OF THE BRANCHED-CHAIN AMINO ACIDS IN THE RATThis thesis is prim arily concerned with the adaptation of leucine oxidation in rats that a re either fasting or have been fed protein restricted d iets. It a lso includes the adaptation o f valine oxidation in rats fed a pro te in -free diet for periods of up to 3 weeks.The in it ia l experiments were concerned w ith resolving the differences of opinion which held in contention the ab ility of the malnourished rat to adapt to a low-protein diet and in particular to conserve the branched-chain amino acids. Much of the disagreement was la te r found to be due to the choice of the radio-active label for measurement o f output following a tracer dose o f the labelledbranched-chain amino acid (B .C .A ). Parallel studies on the f i r s t two enxymes concerned with the catabolism of the B.C.As were also carried out in liver and gastrocnemius muscle to understand better the control mechanism and ch ief s it e o f oxidation. The preliminaiy studies demonstrated dehydrogenase a c t iv ity in skeletal muscle a a well as in liver.It was the f i r s t demonstration of this enzyme ac tiv ity in rat skeletal muscle. Moreover, the degree o f enzyme ndaptation led to the hypothesis that skeletal muscle was the chief site of both oxidation and its control. Further studies were carried cut .o observe the effects of feeding a protein-free diet to rats o f varying ages for periods of 1 , 2 or 3 weeks. Enzyme activities and oxidation were measured. This gave further support to the original hypothesis. Subsequently a more accurate amaaure of■keletal le leucii idati loped. By using the method o f3a constant t a l l r e in infusion of a radioactive tracer in vivo i t was possible to estimate accurately the to ta l body flux o f leucine, together with total body protein turnover and rates o f leucine oxidation. S im ilarly , by giving a constant infusion o f the same radioactive tracer to the perfused hind-limb o f an identically treated rat, i t was possib le to arrive at a more accurate measure o f the contribution o f sxe le ta l muscle to tota l body leucine oxidation.TITLEABSTRACTSECTION I - INTRODUCTION TO THESIS.( j ) Branched-chain amino acida in malnutrition, ( i i ) Adaptation of tha bran oh ad-chain amino acida t o protein deplation.( i i i ) Degradation of branched-chain amino acida. ( I t ) S i t e o f BCA degradation.( T) O xidation of BCAa by muscle.( r i ) Aminotransferases.(▼ li) Dehydrogonasea.( r i l l ) Adaptation in oxidation o f BCAa.( i x ) The perfused hind-limb preparation.SECTION I I - THE D W T OP DIET OH OXIDATION OP THEi dINDEX( * )( i t )(111)(1»)[ » ](1)(u )(111)[c l‘ p l i w i l ' c-i;y».-3 in n v o t 1 - . .t r j . - ie j iu . u . c l . . ( l ) Introduction .( i l ) j[n r l t r o oxidation in o e l l - f r e o extracts.( i l l ) R e su lta .HRANCHKD-CHAIN AMINO ACIDS..t e s s iIntroduction .Anl — l a and diets. M a te r ia ls and method. R e su lts and dlsouseion.P r in c ip le s .Optimum assay conditions R em u lta and discussion.PACE7101113131316313333*33* 1M* 1h iS8ÌI 5 5 nt!lINDEX( i ) Introduction.( i l ) Animala and d lata . ( i l l ) Enzyme assays.( I t ) Results.[ * ] Summary o f Section I I . 52SECTION I I I - LEUCINE TURNOTETl IN THE WHOLE BODY AND IN 5*l ]THE PE-tETJSED UN D -LIMB.Pool. 5*» I 56( 1 ) Introduction. 56( 1 1 ) Methods. 57( 1 1 1 ) Leucine oxidation rates in vivo. 61( IT ) Rates o f protein synthesis. 65( t > Results. 67( t1 ) Total leucine flux in vivo. 71(T i l ) Discussion. 75>1( 1 ) Introduction. 77( u ) The perfused hind-limb. 77( 1 1 1 ) ▼ lability o f the hind-limb preparation. 85( I t ) Methods. 89( t ) Oxidation o f leucine in the hind-limb. 91(Ti> Protein synthesis rates in the perfused hind-limb. 951 Conclusions. 99PAGESB3TI0N IV - DISCUSSION. 105( i ) Enzymes concerned with the oxidation o f BCAa. 105( i i ) The ohoice o f la b e l. 111( i l l ) The DL- or D-isomer in measurements o f oxidation. 115( I t ) Tissues oxidizing leucine. 116(v ) Hormonal effects on BCA oxidation. 117( t1) E ffect o f fasting on BCA oxidation. 119( r i i ) E ffect o f feeding low-protein diets on BCA oxidation. 123( r i l l ) Oxidation rates in v ivo . 125( l x ) Oxidation rates in the h in d -lin t. 126(x ) Leucine as a possible regulator of ske leta l muscle 129protein synthesis and leucine oxidation.SUMMARY 133ICnKJWLEDSB'lENTS 134LIST or FIGURES 135LISP or TABLES 1J6LIST OP SCHEMES 139REFERENCES 140INDEXI N T R O D U C T I O NThe in i t i a l impetus fo r th is work came from the long-standing interest o f th is ttiit in protein energy malnutrition in children (Waterlow, e t a l I960; Water l ow & Alleyne, 1971). Hie work was primarily concerned with examining the metabolic and biochemical changes produced by protein malnutrition. Protein malnutrition, when i t reaches the stage o f c lin ical i lln ess , represents a breakdown o f adaptive mechanisms (Waterlow, 1968). Thus, as Waterlow <■ Alleyne (1971) pointed o u t, progress in detecting and preventing protein malnutrition depends ultimately upon a better understanding o f adaptive changes at the le v e l o f both the whole organism and the c e l l .There i s a v ide clin ical spectrum o f malnutrition from the predominantly oe de matous child with Kwashiorkor to the wasted marasmic child. In atten çts to understand these extreme forms o f malnutrition and the interm ediate stages o f the disease more attention is now being paid to the s i& iific an ce o f fundamental biochemical changes. Definition o f those changes which represent breakdown o f the adaptive mechanisms is clearly important (Waterlow, 1968).Animals a re ab le to survive long periods on diets in vhich protein is reduced o r omitted ( Men des & Waterlow, 1958). This capacity to survive suggests the presence o f mechanisnm fo r adaptation which lim it the effects o f a lte red nutrient intake on metabolism and thus on the composition o f the body. The process o f adaptation may need to bedistinguished from the responsiveness o f the body to short-termchsnges in nutrient supply. This d istinction relates not ju st to the tiBM-period over which adjustments occur in relation, for example, to food supply, but perhaps also to the mechanisms which are brought into p lay . Thus there is a great deal of evidence to suggest that very short term responses occur in the hepatic metabolism o f amino—acids in the few hours a fter a meal. In the rat an influx o f amino acids leads to a very rapid increase in ornithine - £ - transaminase| the enxyme returns promptly to thepre-feeding levels within 24 hours (Kaplan ft P ito t , 1970). However, i f protein is withheld from a rat's diet for a period of days, then further changes in hepatic protein metabolism occur which tend to maintain body stores o f protein even though the capacity to respond to a sudden inflow o f amino acids is reduced (Schimke, 1962). Whereas the immediate response in ornithine - J - transaminase is probably stimulated d irec tly by the concentration o f amino acids in the tissues, the slower adaptive response may depend not only on the level o f an amino acid but a lso on the circu lating level of insulin or other hormones. These examples are given to illu s tra te the problem of d istinguishing between responsiveness and adaptation* no claim is made, however, that there is an absolute distinction between the two processes. Moreover, i t i s probable that adaptation includes not only changes in hormonal pattern, but also longer term effects on body composition.The processes o f adaptation obviously depend on the nature of the change in d iet and perhaps on whether a deficiency in body stores or a change in function has already developed. Thus i f the animal is deficient in protein but receives s diet adequate in a l l respects, other than protein, one would expect the adaptation to beprimarily with the conservation of body prote in . This somewhat teleological argument is borne out experimentally» rats or Man fed a low protein d ie t show an in itia l rapid decline in the excretion o f urinary nitrogen, followed by a slower decrease until the nitrogen output reaches a steady minimum or 'endogenous* level (Munro, 1964). This low protein diet leads to a series o f changes in tissue protein metabolism which vary considerably from one organ to another. In short-term experiments, very la b ile organs, such as liv er , pancreas and smal 1 intestine are major contributors (Munro, 1964) to the excretion o f H in the u rin e , whereas in more prolonged experiments muscle becomes a major source o f protein (Mendes A Vaterlow, 1958» Vaterlov A Stephen, 1966). Eventually, the proportion o f the in i t ia l protein lost from muscle may approximate or exceed the percentage lo st from liv e r and other more la b ile tissues.The eventual e f fe c t o f the adaptation is a redistribution o f body protein. The major lo ss is borne by muscle and skin ( in the ra t ), while the protein content o f essential tissues such as brain, myocardium and kidneys, is re la tive ly well preserved. Hormones probably play an important part in bringing about this red istribution .Starvation involves d iffe ren t metabolic processes o f great importance in re la tion to amino acid and pro te in metabolism (C a h il l , 1971). There is a -ed fb r glucose as a substrate for certain tissues, eg. brain, which is generated by gluconeogenesis from amino acids (Harper, 1965). Thus in starvation the amino acids serve an additional function to that involved in protein metabolism per se. and there is a greater breakdown and a higher rate o f nitrogen excretion in the■ine than occurs in the protein depleted animal (Srhimke, 1962).10jij Branched—cha i n amino ac i«la in malnutritionOne aspect o f the adaptive mechanism» which has been investigated in this thesis is the metabolisa of branched-chain amino acids and the relevance of this approach to malnutrition must firs t be considered. In 1963, Holt and his co lleagues first showed that the total amounts o f free amino acids in the plasma of malnourished children are one h a lf of the norsial value and tha t there was a d istorted pattern o f individual amino acids in ch ildren with Kwashiorkor. The levels of the branched- chain amino acids were markedly reduced whereas the concentrations of lysine, histidine and phenylalanine were l i t t l e changed. In contrast, there was a rise in the concentrations o f some non-essential amino acids. So conaistent d id this finding appear to be that Vhitehead (1964) developed a sim ple test for protein deficiency by assessing the degree of distortion and expressing the resu lts as a ratio o f selected non-essential to e s sen t ia l amino acids in the plasma (the N«E r a t io ) .Thus the greater the d is to rt io n the higher the NiE ratio (Vhitehead,1969). There is general agreement (Vhitehead ft Dean, 1964| McLaren,1965! Viddowson ft Vhitehead, 1966) that the amino acid ratio is usually elevated in patients w ith Kwashiorkor, but not in a l l malnourished children. An in fection o r a diet low in ca lo rie s reduce the distortion and return the ratio towards normal. The intake o f protein immediately before the te a t also proved to be important since the ratio returned to normal w ith in 1 - 2 days of refeeding the child su ffe rin g from Kwashiorkor (Itty e rah at a l, 1965). This suggested that the amino acid ratio is more representative of the extent to which protein intake is lim iting than of the s ta te of protein deficiency , per se. A simple state of protein depletion o f body tissues could not be the key factor in causing11distorted amino acid levels , since sarasnic children have a markedf a l l in the protein content of the body but the amino acid ratio remains normal. Conversely, a d isto rted amino acid ratio can be produced in healthy adults after a short period of 2—3 days on a low protein diet, before any appreciable loss of body protein occurs (A lleyne * Picou, 1971). Nevertheless, the ratio haa been used in f ie ld studies to detect marginal disturbances in protein metabolism . Thus it seesis not treasonab le to think o f changes in branched-chain amino acids as e ith e r significant markers o f the disease process or ss intimately involved in the progression of the disease.Adaptation of «he Branched-chs n amino acids to protein depletionMost studies on adaptive ensyme changes have been made on liver. However, both in man and in the rat the amino acids whose concentratic-s in plasma and tissues are most decreased in protein deficiency are the branched-chain amino acids (BCA s ) , p a rticu la rly valine (Whitehead ft Dean, 1964). According to M ille r (1962 ), the extra^hepatic tissues are as capable as the liv er o f oxid is ing leucine whilst the findings o f Mortimore ( 1970) suggest that valin e is not oxidised at a l l in the l iv e r . The lowered levels of BCA s in plasma seen in protein depletion suggest an inability to conserve these amino acids (Table 1 ) . However,Me Far 1 an e ft Von Holt (1969 a) showed a decreased oxidation o f Dt.[2^c] leucine in the rat ( in vivo) fed a pro te in deficient d iet for eight weeks. In view of these findings i t seemed of interest to investigate in more detail the adaptive capacity o f the ansymes which catabolise branched-chain amino acids, not only in l iv e r but also in extra- hepatic tissues (skeletal muscle). In comparison with liv e r , very l i t t l e is knowsabout adaptive changes in muscle encymes!12TABLE IAaino Acid leve ls in plasma o f rats fed d ie ts o f di fferentNDptE ra t io * and the observed N:E raitio in each group of ratsDIET 0 lONDp.-E(P*0 035” NIJp : B oles/ml plasma)0 CO SlipAMINO ACIDASPARTATE 32.8 49.3 73.7THREONINE 90.2 43.6 69.6SERINE 313.0 437.0 694.0ASPARAGINE TO.O 49.8 43.8GLUTAMATE 229.0 244.0 279.0OLUTAMINE 679.0 781.0 901 .OOLTCINE 236.0 320.0 472.3ALANINE 840.0 802.0 923.0TALINE 233.0 163.0 94.7ISOLEUCINE 93.9 64.9 39.2LEUCINE 148.0 119.2 81.3TYROSINE 84.4 30.8 18.8PHENYLALANINE 48.7 42.3 39.4METHIONINE 118.0 6 8 .2 2 0 .2HISTIDINE 86.0 99.1 130.0ARGININE 69.9 107.0 76.7LTSINB 824.0 410.0 362.0A ll rats weighed 65*Vhitehead‘ s N:E ratio 2.78 4.89 7.87Ratio o f energy supplied by u t il is a b le protein : to ta l Metabolisable energy.Ratio of defined non—essential amino acid i essential aaino acids.Non East ALANINE I m LEUCINEGLTC1NK ISOLEUCINESERINE PHENYLALANINEGLUTAMATE VALINEGLUTAMINE TTROSINETHREONINE METHIONINEVhi tehead NiE ra tio130*0 Degradation o f Branched—cha i n amino acidsThe metabolic pathways from the BCA a to the ir fin al products acetyl CoA, acetoacetate and succinyl CoA have long been elucidated (Meister, 1965). The s te p s involved in the conversion o f leucine, for example, have been id e n t if ie d by isotopic experiments and more recently by enzymatic s tu d ie s . A ll the BCA s in i t ia l ly fo llow a sim ilar pattern, i .e . transamination to their respective e* - oxo acid, followed by ir r e v t « » ''6'* oxidative decarboxylation to the corresponding acyl CoA d e r iv a t iv e . The remaining steps are analogous to those o f fa tty acid ox id ation . The decarboxylati on step is not reversible! this is com patible with the in a b ility o f animals to synthesise the BCA s from intermediates other than the analogous • i - o x o acids. The scheme (Scheme I ) for the degradation o f leucine illu stra tes the well-known ketogenic properties o f th is amino acid. Although the degradation o f valin e in it ia lly proceeds by a series o f reactions sim ilar to those involved in leucine catabolism (Scheme I ) the end products are qu ite d if fe re n t , since valin e 1% glycogenic (Rose et a l , 1942). Iso leu c ine is ketogenic under certain conditions, but under others leads to the formation of carbohydrate. The significance o f the BCA s fo r the synthesis o f cholesterol has also been established (Symposium on Cholesterol Metabolism, 1955).( » ) a i t , . f BCA P~,r.'la l ionThe BCA a have long been considered unusual among the essential amino acids in that catabo lism was presumed to occur predominantly in extra-hepatic tissue. This view was supported by resu lts obtained by liv e r perfusion studies and on the eviscerated surviving rat (M ille r , 1962). M ille r showed that the extra-hepatic tissues o f the1*»SECTION I I B ( i )SCHEME I!« «c t io n » involving th> radiosc♦ irely labelled carboxyl group in I .f l ’ V j 1 ynr i noCIS -Lour in. « ( -o x o -f lu ta rtt* Mi no transferase [M .C.2 .A . « . * . )T -■ >*COOH«( -oxo-isocaproate dehydrogenaseL -o io -n o riD roa t •1 itatively by saturated carl sulphate in(A ) Reaction halted by ,2N K SO expelling — C02iS solutionI80-VALERTL CoA- C - S CoAJ ACETOACETATE] * [ ACKT1L CoA15eviscerated surviving rat could oxidise DL^t^c) leucine u e ffe c tive ly ss the isolated perfused liv e r . There ar* however, several objections to his experimental protocol. The rats were starved for 16 -18 hours before the operation and dilu ted blood was used as the perfusate. Therefore the liv e r entynes may have adapted to starvation conditions. ^jrthermore, no allowance was side for d ifferences in intra ce llu la r specific radio*activity that might arise through the administration of the same amount of DL (l ) leucine in preparations o f very different weights (eg. liv e r vs eviscerated ra t ). This makes i t d i f f ic u lt to draw any firm conclusions from the data, except that liver and extra-hepatic tissues do oxidise M .[l14C Jleucine.(V) Oxidation o f BCA s by muscleSeveral groups of workers had suggested that muscle night be the main s ite o f BCA oxidation before this thesis was begun. Their evidence was based on the demonatration o f '^CO^ production from radioactively labelled leucine in isolated perfused heart (Clarke, 1997) or incubated diaphragm (Manchester, 1969).Young (1970) also pointed out that skeletal snjecle has the highest total BCA transaminase activ ity o f a l l tissues in the rat, being approximately lOO tines the amount o f enayme in the liv er . He concluded that the catabolism o f the BCA s took place mainly in muscle. Rlwyn's (1970) voih supports this view! for most amino acids the greater part of the load absorbed into the portal vein following a protein meal is metabolised in the liv e r and the amounts transferred to the peripheral blood are smalli the BCA s are exceptions in that a larger proportion of them pass into the general c ircu lation , presumably16being trails am inated in muscle and kidney.In sp ite of these stud ies, u n t il the present thesis was begun no quantitative evidence had been produced to support the hypothesis that skeletal muscle was the m ajor s ite for BCA oxidation. Moreover, quantitative measurements of t o ta l body leucine oxidation and of leucine oxidation by individual organs had not been attempted.Although a great deal of work had been carried out on the BCA transaminases and dehydrogenases, the ia su e was confused by the diversity of choice o f the tissues or animals studied. A short review o f the knowledge up to that time w i l l i l lu s t r a te this point.(Vi) Aminotransferase«Iehihara et al (1966) and T ay lo r A Jenkins (1966) f i r s t isolated and characterised a specific branched-chain aminotransferase [E .C.2. 6 .1 .6 .) in Hog Heart . Subsequently, three types o f ami not ransferases ( isosymes 1, I I and H I ) were repo rted in various tissues o f the rat and hog (Afci a l , 1968| Aki et a l 1969» Ogava et a l , 1970). These isosymes could be distinguished e ith e r by DEAR ce llu lo se chromatography or by immunological techniques. The properties o f these ensymes are summarised in Table 2.In the hog there is no evidence o f isosyne I I (A k i, Tokojina A Ichihara, 1969) but isosymes I and I I I are widely d iatribu ted . Ogawa et a l , 1970) have shown that isoayae I I I iB only found in the supernatant fraction o f hepatoma c e l ls . In the rat, Aki, Ogawa A Ichihara (1968) showed that isosyme I was d is tribu ted evenly between the supernatant and mitochondrial fr a c t io n s of the liv e r . I t transaminated a l l three BCA s and had a r e la t iv e ly low km. Ieosyme I I , however, was found exclusively in rat liv e r , m ain ly in the mitochondrial fractionTABU 2MI CHABLIS COWTANTS OP BRANCHED-CHAIN AMINO TRAXSPERASES k DEHTDROGENASESDonor AsinoAcid Source M « ) pH ReferenceCentral branthed-chainaaiaotransferase Lenóne Pig Heart 3.» )▼aline Pig Heart 11.0 j 8.6 Ichibara A Kovam» (1966)Isoleucine Pig Heart 3.» )1soave» I Lenóne Rat Liner 0.73 )▼aline Rat Lioer 4.J0 » 8.2 Aki. Ogava è Ichihara (1968)Iaolenóne Rat tirar 0.84 )Isoajne II Lenóne Rat Liner 29.0 )▼aline Rat Liner )) 8.7 Ahi, Ogava k Ichihara (1968)Isolaneine Rat Liner -Isoayne III Lenóne Ho* Brain o . w▼aline Hog Brain ' ■* 8.0 Aki, Tokojina k Ichihara (1969)Isolaneine Hog Brain 0.67BCA Aninotransfera»» Lenóne Rat Liner 1.70 7.0 Presont vorkLene ine Rat Musei» 0.37BCA Dehvdrogenase Lene ine Rat Liner 0.17 )) 7.0 Present vorkLancine Rat Musei» 0.17 )Uncino Rat Liner 0.20 ) 6.8 Volhueter è Harper (1970)1Band had a high km fo r leucine) the other BCA s showed l i t t l e or no ac t iv ity with the emyse. Induction o f iio iy se I I was observed mainly in the supernatant fraction . Isosyme I I I was almost exclusively in the brain . Is*» cymes I and I I I have quite sim ilar properties and can transaminate a l l three branched-chain amino acids at approximately equal ra tes . The km o f isosyme I for valine ia considerably higher than fo r leucine and isoleucine, the values fo r which are about the same ( Aki et a l , 1968). Krebs (1972) has discussed the p rinc ip le of control o f BCA degradation through km, as f i r s t suggested with special reference to amino acid metabolism by M allette, Exton k Park (1969). Thus, i f the km values are in *;en»r»l high compared to concentrations o f free amino acids, then any increase in anino acid concentration in blood and tissues automatically causes an increased rate of amino acid degradation. However, in i l lu s tra tin g this po int, Krebs gave km values fo r the isosyme I found in hog heart. He then proceeded to study the e ffe c ts o f a protein -free d iet on rat liver transaminases, which elsewhere have been reported as having low km values (Aki et a l , 1968).No reports are availab le to suggest that rat skeleval muscle transaminases have the same km values as those described in hog heart. In fa ct , in the present studies ( Table 2 ) the km value for leucine wasO.lTiH in rat skeletal muaclei this is an order of magnitude lower than the values taken by Krebs to represent skeleta l muscle transaminases in the ra t . However, the princ ip le remains, that control by km is a "fine contro l" mechanism. There is an additional "coarse control” brought about by adaptive adjustments o f the eniyme capacity, through variations either in the rate o f ensyme synthesis or in the rate o f19Studies on tiie adaptation of branchod-cliain aminotransferases have been almost exclusively in rat l i v e r and kidney. Shirai et a l , (1971) found that isozyme II was more responsive to induction than isozyme I in liver. Isozyme II had a shorter half l i fe and was rapidly induced by cortiso l, high p-otein fe ed in g , and gluconeogenic conditions. Ildzyme I in liver was not affected by any of these. The kidney f&ezyme I was induced only after continuous administration of co rtiso l over 7 days; hypophysec tomy also induced the enzyme whilst adrenalectomy decreased it . Since McParlane * Von H o lt (1969 b) had shown a greater proportion o f their enzyme preparation in liver to he in the mitochondrial fraction, they were probably studying f$ozyme I I . However, although they showed that low protein feeding induced an adaptive response, this was only observed in the mitochondrial and not in the supernatant fraction.Mimura et al (1968) studied the transaminases in rat skeletal muscle, as well as liver and kidney. T he ir measurements showing induction in both muscle and liv er transaminase activities by a protein-free diet were in direct c o n f lic t with the reduced enzyme activ ities of liver BCA transaminases in protein depleted rats reported by McParlane 8 Von Holt (1969 b ) . Mimura et a l also found that after administration o f hydrocortisone for 3 days, BCA transaminase activity was elevated in both l i v e r and muscle. The activ ity o f BCA transaminase was greater in muscle than liver. These workers did not distinguish which isozyme was b e in g studied but since the preparation was equally reactive with a l l three BCA's this would suggest that they wei v in fact looking a t Isozyme I. Again this is in conflict with the work of Shirai 8 Ich ihara (1971 ) vho found no effect o f cortisol om usyse I in rat l i v e r i they did show that kidneyBCA transaminase (itozyme I ) induced but.soadministration of cortisol.The picture, therefore, at the time when this work was begun, was rather confused. Perfusion experiments suggested that BCA s are siostly oxidised in extrahepatic tissues. Nevertheless, the majority of studies on the ac tiv ity o f BCA transaminases (the enzymes which initiate oxidation) had been made on liv e r . A protein- depleted d iet was found to cause an adaptive f a l l in one o f the liver enzymes (isozyme II , McFarlane ft Von Holt, 1969 b) whilst a protein- free diet was found to cause an increase in the liv e r enzyme (isozyme I , Mimura et a l, 1968).The conflicting evidence reported above showed an obvious need to investigate the BCA transaminases in several tissues, particularly since there was direct conflict not only on the reported effects of protein-depletion on BCA oxidation in vivo but also on the manner of adaptation o f the transaminases in rat tissues.6**9 DehydrogenasesTransamination of the three BCA s leads to the fo llowing 4 -o x o acids on which the dehydrogenases react t-leucine -* « - oxo-isocaproate ( st KIC) valine -ft« - oxo-isovalerate ( « KIV) iaoleucine - » « - oxo-methyl valerate ( sc KMV )Danner ft Bowden (1966) demonstrated separate BCA dehydrogenases in intact rat l iv e r mitochondria . A preliminary note reported the existence of separate dehydrogenase mechanisms also in human and bovine leucocytes (Ooedde, Hufner et a l , 1967). Connelly et al (1968) then isolated a partially purified enzyme from bovine l iv e r cytojlarr: inactive towards « KIV but active with «KIC and «KMV. This was21ten tative ly called an at KICj at KMV dehyclrogeiiu.se. The dual spec ificity o f th is preparation could be accounted fo r in two ways. E ither it contained two d ifferent enzyme complexes that were purified in p a ra lle l, or there existed one enzyme complex which had both a c t iv it ie s . Bowden ft Connelly (1968) were able to demonstrate a single enzyme complex by physical, chemical and k inetic treatments. They also demonstrated separate el KIV dehydrogenase activ ity almost exclusively located in the mitochondria. The former single enzyme complex was distributed equally in both supernatant and mitochondrial fractions of bovine liver. Volhueter A Harper (1970) were unable to d istinguish separate dehydrogenases in the rat liv e r mitochondria as attempts to pu rify the enzyme were rather unsuccessful. Earlier work by McParlane ft Von Holt (1969 b) also demonstrated BCA dehydrogenase ac tiv ity in rat liv e r mitochondria, but because of the lack of evidence of d istin ctive BCA oxo acid dehydrogenases at that time,they were reluctant to attribute the ac t iv ity to a specific enzyme. Nevertheless, in view o f more recent work we may conclude that they were actually measuring the dehydrogenase in rat liv e r mitrochondria. Interestingly, their work suggests the p o ss ib ility o f two separate enzymes located in the mitochondria and cytoplasm.Both Connelly et a l (1968) and Volhueter and Harper (1970) looked at dehydrogenase d istribu tion in various tissues of the ra t and other anisutls. In the rat, the greater part of the ac tiv ity was to be found in tli e liv e r and kidney while in beef the ac tiv it ie s were more evenly distributed throughout l iv e r , kidney, heart and skeletal muscle. At the time this thesis was begun (1972) no ac tiv ity o f the dehydrogenasehad been found in rat skeletal22(s/lii) Adapta 11 on in Ok id « t ion o f BCA sBefore 1972 only « few workers had studied adaptation in BCA oxidation. McParlane k Von Holt (19W a) demonstrated a steady decrease in the oxidation o f DL(214c ] leucine in r a t s given a diet containing 2Og protein/kg fo r eight weeks. This d e c lin e accounted fo r a 75^ f a l l in oxidation fron week two to week e ig h t . Neale (1972), however, was unable to show any adaptation in the oxidation o f U ,4C labelled branched-chain aaino acids in r a t s given a diet containing lOg protein/kg for 2 weeks. Both these investigations nay be c r it ic ised because o f the choice o f label| th is w ill be discussed in detail at a la te r stage.Further studies l^ y McParlane k Von Holt (1969 b ) were undertaken to investigate the site o f control of this ox idation . They concluded that there was a block in the decarboxylation o f le u c in e with a reduction in the aitochondrial transaainase and D-amino acid oxidase in liv e r . Their work also included measurements o f the BCA ■*< KIC dehydrogenase, despite their unwillingness to recogn ise th is . They showed the p o ss ib ility o f two separate ensymes loca ted in the a ito - chondrial and cytoplasmic fractions from liver homogenates; the aitochondrial ensyae was decreased in protein depleted rats but the cytoplasaic ensyae reaained unaltered. The reduced Mitochondrial ensyae ac tiv ity led to an increase in d - o x o -iio c ap ro ic acid ( st KIC) in the aediua. Ihis was reflected in vivo by an increased urinary output o f the branrhed-chain « . oxo acid. Despite t h i s evidence, it was d i f f ic u lt fo r then to suggest an absolute decrease in dehydrogenase ensyae ac tiv ity since they found no significant d i f fe r e n c e in the oxidation o f labelled A KIC by rat liver mitochondria from control or protein depleted rats .I A Y T T u a m i â v a a â i a i2kacid flux . Similar data can fc2 (Manchester, 1965» Toung, 1970) it seemed paradoxical that the BCA dehydrogenase had not been demonstrated in th is t issu e (Volhueter ft Harper, 1970) at the time when this thesis was begun. Moreover, many workers were s t i l l pointing to the liver as the ch ief organ in which the BCA's are oxittaed when evidence was accumulating to suggest that muscle was the major site (M iller, 1962» Toung, 1970» Elwyn# 19 70 » B1 oxaa, 1971 ) . With a modification of the method o f McParlane ft Von Holt (1969 b) and with gentler homogenisation o f muscle tissue, el KIC dehydrogenase activity was observed in the gastrocnemius, extensor digitorum longus (EDL), soleus and p la n ta r is muscles o f the rat (Table 3 ). furthermore, as discussed in i s c t io n I I C because o f the rapidity with which the muscle dehydrogenase a c t iv i t y adapted in times of dietary stress, before any adaptation warn observed in the liv e r ensymes, it was necessary to use the hind-limb preparation to study the mechanisms involved in adaptation and to quantitate the contribution o f skeletal muscle to the oxidation o f leucine in the whole body. In paralle l experiments, total body leucine o x id a tion was determined in vivo by the constant intravenous infusion of a t ra c e r dose of radioactive L ( l * * c j - leucine.27THE EFFECT OF DIET OM OXIDATION OF THE BRANCHED CHAIM AMli-'O ACIDS.A The effect of 1ow- trotein feeding and atarvatlon on theoxidation of DL- and h-lsomera of leucine in v ivo .( i ) INTRODUCTIONThe rate o f leucine oxidation measured in vivo by monitoring the excretion o f 1^CO? a fte r a s ingle intraperitoneal tracer dose o f Dl [ 2 1*c ] leucine, has been shown to decrease in adult rats fed a low-protein d iet (2 0 g casein/ic«) fo r 8 weeks (HoFarlane A Von Holt, 1969 m). The authors suggested that the decline in oxidation rate resu lted fro * a block in the decarboxylation o f K-oxo-isooaproio acid in response to a reduced ac t iv ity o f the leucine i H -oxo-glutarate aainotraneferase enzyme. This was confirmed by enzymatic assays o f mitochondrial fraction s prepared from the l iv e r (Me Far lane A Von Holt, 1969 b ) .However, Meals (1971 )* working with uniformly la be lle d 14C amino acids in the L-isomer form only, fa iled to demonstrate any conservation o f either leucine or valine when in jacted intravenously o r intrm gastrioally into protein-depleted weanling rats . Because o f these discrepancies both in methodology and resu lts, l [ i 14c J -leuc ine was chosen to study the effects o f protein depletion on leucine oxidation in vivo. This ohoiee, discussed extensively in Section I V ( l i ) , a lso allowed a comparison with the in v it ro data . Measurements were also made withfoL 114c ] leucine because o f the possible involvement o f the D-aaino acid oxidase [ l . C . 1 . 4 . ) . } ] and to document the degree to which the differences between the findings o f Me Far lane A Von Holt and Neale might resu lt from the choice o f la b e l.28A ( i i ) ANIMALS AND DIETSPénale hooded rata (Animal Suppliers (London) Ltd) weighing 35-40g were housed three to a cage and allowed free access to a powdered d iet which contained (g/kg) I - Casein (Casumen t Prideaux Milk Poods, Bvercreech, Someraot) 109» maize starch (Corn Products Ltd, Manchester) 426» dextrinized starch (Corn Products) 272» solka floe (ce llu lose ) (Johnson, «Jorgensen ft Vettre Ltd, London) 91» arachis o il , 45» mineral s a l t , 45» B-vitamin mixture, 10| fat soluble vitamin supplement, 0 .9 and L-methionine (Sigma Chemicals Ltd, London) 0.9. See a ls o Table 4.Details of the mineral salt mixture, the B-vitamin mixture and fat-soluble vitamin supplement are given by Payne ft S tew art (1972). The ratio of energy supplied by u tilizab le p ro te in to the total metabolizable energy (NDp:E ra tio ) was C iO fcontrol d i - t ) and maintained growth at the rate o f 3.0g/d.A low-protein (L .P ) diet was designed to maintain the an im als at a constant weight over a 2 week f e e d ip e r io d » the appropriate NDpxB ratio for this was found to be O This diet was the same as the control (H .P) diet except that it contained 3 2 .9g casein/kg, the difference being replaced by an equivalent weight o f s t i ie starch (Table 4 ) .A ( i i i ) MATERIALS AND METHODA ll radioactive materials were obtained from the Radiochemical Centre, Amersham, Bucks. Specific radioactivities of amino acids were 1 l [ Í 14cJ-leu cine (60 mCi/mmol), L [U, 4CJ-leucine (lOsCi/mmol) and DL [l ,4C Jleucine (IO mCi/mmol ).SECTION IIÀ(ii) TABLE ACOMPOSITION OP EXPERIMENTAL DIETSLow Protein (L .P ) (0.035 NDp:K)Zero Protein (0.00 NDp:E)( t ) <«> (ft)Maize Starch 2345 2773 2950Dextrinized Star.:h 1500 1500 1500Casein 600 175 -"Solka PI oc* (c e l lu lo s e ) 500 500 500Arachia o il 250 250 250J ft P s a lt auxture 250 250 250Vitamin B group 55 55 55L-methionine 5.0 2 .2 -Pat-soluble302,5 Diphenyloxasole waa obtained from Koch-I,ight Laboratories Ltd, Colnbrook, Bucks.Amino acids were from Sigma Chemicals, London.A l l other reagents were from B ritish Drug Houses, Poole, Dorset.Measurement o f Catabolism of 14C-labelIed Branched Chain Amino Acids IN VIVO by Pulse Intragas trie Dosage.Animals were allowed free access to food and water until 5 hrs before testing. In th is way, the stomach would be empty before the experiments began. The oxidation rates o f various 'Relabelled branched—cha» n amino acids were then assessed by the intragastric in jection of the label in a solution o f NaCl (9g/ l, pH7.0 yjci per lOOg body-weight) with the appropriate branched chain amino acid as ca rrie r ( l /Jmol*L-leucine/lOOg body-weight).Accurate measurements o f syringe weights before and after injection were used to determino the amount o f label given to each animal - The animal waa then placed immediately within a sealed glass container and expired was trapped by drawingthe expired a ir through three tubes in series , each containing 40ml 4M-NaOH.Trapping was considered to be complete, because the third tube contained less than IjC o f the trapped labe l. Collections were ■«ade for 3 hours because McFarlane A Von Holt (1969) observed that the percentage o f the dose excreted as ,4C<>2 reeches a p lateau by 3 hours. Aguilar, Harper A Benevengai1972) also observed the constant production o f , 4C<>2 a fte r the 3-hour time p eriod with many o f the amino acids. Valine, however, showed a s ligh t decline in production a fter this period which>ly or increased demandthought to indicate a limited31in other metabolic pathways.The NaOH from the 3 tubes was pooled and 1 ml sample ac id ified with 2 ml 2M.HgS0|i in a Marie-Flask. Evolved ^OOg was trapped on a f i l t e r paper soaked with 0.25 ml phenylethylamine in a centre w ell. The f i l t e r paper was then counted with 13 ml o f a 3:10 methanol/toluene mixture containing O.Uji P.P.O (2 , 5 diphenyloxazole) in a Packard 2U20 liqu id sc in tilla tion counter.A ( iv ) RESULTS AMD DISCUSSIONIn vivo oxidation o f Dl £i ***cJ leucine was greater than that o f leucine in a l l groups except the animals on the control d iet who were fasted fo r U8 hrs. Fasting produced a marked increase in the evolution o f ll*00g except in the rats on the control diet tested with D L ^l^cJ leucine, and fasting o f the protein-depleted animals restored the output i l ’c . t to the le ve ls found in the control rats without fasting (Table 5 ).Oxidation o f both DL £ l***c j leucine and L laueina wasreduced in the animals given the low-protein diet.The reduction in EL-leucine oxidation with low-protein feeding,seen by McFarlane & Von Holt (1969 a ) in adult rats , has beencon firmed with young growing animals. The use o f the L-isomerhas a lso shown that the results re flec t changes in the normalleucine catabolic pathway and not singly alterations in the activityo f D-asiino acid oxidase E.C.l.fc. 3.3. The observations madeby Neale (1971) with U ^C labe lled leucine and valine in which he fa i le dto find evidence o f adaptation, may have been due to several pointsin his experimental procedure. F irstly , we do not know whether thediets were isocaloric and hence the animals could have been exhibiting¡XTABU 5In vito oxidation of [ 114C]-leucine usine either the X - or L-isoner in rnts given a high- or low-protoin diet or fasted. (Mean values with standard errors where given)Isoaer in groupXDp:E ratio of diet*Whether fasted for 48 hrEvolved '*C0j « of dose given)DL 2 10 - 31.32 10 ♦ 27.03 3.3 - 1 1 .1 - 0 .1 a3 3.3 ♦ 22.0 t ) .2 aL 3 10 - 13.3 - 0.3 to3 10 ♦ 23.0 - 3.2 c3 3.3 - 4.0 1 1 .0 b2 3.3 . 10.3S l.t i . lK . l c -f .r i.o n of ,roap, values narked with the saaw letter differ significantly at thefollowing levels i a, P {0.01{ b. P{0.001; e, P {0.02.Percentage of total netabolnable energy supplied by utilisable protein.•p«*wj jo t*tp ot»\oj4-»o\ JO -**i* « uoaiI ()«J ai Jiwti-qForcontofc of total attoboliMblo *n*rgy supplied by utilisable pretti33the pattern o f oxidation Been in fasting rats (Table 5)« Secondly, 14C02 excretion was measured a fte r only 1 hour, at which tine constant production of 14C02 has not been attained (A gu ila r, et a l. 1972). Finally , the use o f U14C labelled branched-chain amino acids fo r this particu lar study is in question and w ill be discussed elsewhere (Section IV ( i l ) ) .H^n^tr-ansferaee K.C.2,6.1,6. and* -oxo-ieccaproate( 1 ) PrinciplesThe princip les o f the assay are shown in Scheme I . Amino acids labelled in the carboxyl group w ill resu lt in labelled A-oxo acids. Subsequent decarboxylation by the dehydrogenase w ill y ie ld 14(X>2. Therefore the amount o f 14C02 plus any labelled si -oxo acid not decar boxy lated w ill be a measure o f the transaminase activity . In the presence o f ceric sulphate (Meieter, 1952),^-oxo-acids undergo quantitative decarboxylation. At a given substrate concentration the ensymioally produced 14C02 from amino acids labelled in the carboxyl group reflects the dehydrogenase aotivlty (A ) and the sum of the ensymioally liberated plus oerio sulphate liberated 14C02 (A ♦ B) reflects the aminotransferase activ ity . These princip les, f i r s t described bF McFarlano A Von Holt (1969 b ), allow the separate estimation of the ac t iv it ie s o f th iA -oxo acid dehydrogenases and aminotransferases .Leucine and valine X-oxo—glutarate aminotransferase activities were measured in both liv e r and muscle by a modification o f the assay system described by MoFarlane A Von Holt (19«9 b ). Both»liv e r and muscle were homogenized at 4°C by hand in a Dual glass homogenizer. Hand homogenization was necessary because preliminary work showed that dehydrogenase ac tiv ity was destroyed i f more vigorous techniques were used. For example, Wolhueter, et a l (1970) were unable to demonstrate sign ificant amounts o f the dehydrogenase ac tiv ity in skeleta l muscle despite the findings o f several workers showing that leucine was oxidized in the peripheral tissues (M ille r , 1962| Manchester, 1970). In the ir investigation on dehydrogenase ac tiv ity the Polytron was used, which is known to disrupt several enzyme complexes. As the dehydrogenase is thought to be oomplexed to several oofaetors (Connelly, Danner A Bowden, 1968) and located on the outer wall o f the inner mitochondrial membrane (Johnson A Connelly, 1972) i t was important to investigate other methods o f homogenization and re-examine the p o ss ib ility o f dehydrogenase ac tiv ity in skeleta l muscle.B ( i i ) Optimum ass-iy conditions were assessed fo r both aminotransferase and dehydrogenase ac tiv ity in l iv e r and muscle, with respect to the ir pH maxima, substrate concentration o f leucine o r valine, and sC-oxo-glutarate and oofaotor requirements (P ig s . 1 -4 ). For muscle fin a l concentrations o f 10maol louoine/f and 15mmol ^ -oxo -g lu tarate/ f were used at pH 7*0 in 25 mM Sorensen's phosphate b u ffe r ( disodium hydrogen phosphate, 25mmol/t, adjusted to pïl 7 .0 )| fo r l iv e r the same system was used exoept that the fin a l concentration o f ^ -o x o - glutarate was 10amol/t.In the f i r s t stage o f the assay (Scheme I ) the 1*C02 evolved (A ) was taken as a measure o f the dehydrogenase ac tiv ity o f the crude homogenate system. Counts were proportional to homogenate concentration35and lin e a r f o r the 60 min. period o f assay at 37°C. Allowance was made fo r the non-specific evolution o f 14C02 from l [ i 14c) leucine on the ad d it io n o f 2M-HgS0^ at the end o f the incubation by routinely incubating blanks and subtracting th is from the experimental values. A f t e r centrifugation o f the incubation mixture at 3900g fo r 10 m inutes, 1 ml o f the supernatant fraction was assayed fo r residual •*-oxo-acid by chexical decarboxylation with saturatedoerio su lphate in ZM-H^SO^. Any non-specific evolution o f 14C02 from the a c t io n o f saturated oerio sulphate on l [ i 1*c] -leuc ine was again ro u t in e ly measured in blanks at th is stage. In summary, then, the p re-assay mixture containing l [ i 14cI leucine, 4 -oxo-glu tarate and Sorensen*s phosphate txiffer a t pH 7.0 was pre-incubated in a Marie fla s k a t 37°C fo r 5 « in . The reaction was then started by the addition o f crude homogenate and halted 60 min. la te r by in jecting 2 ml 2M H2S04 through the rubber cap o f the fla sk . 14C02 evolved was trapped in 0 .2 5 i l phenylethylamine soaked in f i l t e r paper in a centre w e l l . A fte r 1 hr. the oontents o f the centre w e ll were transferred to a s c in t i l la t io n v ia l and counted in the manner described in Section I IA ( i l l ) . The incubation mixture was then assayed as described above fo r residual 14C 4-oxo-acld and 14C02 trapped and counted in a s im ila r manner as before.RESUM-S AND DISCUSSIONB ( i l l ) Determination o f optimum assay conditions fo r leucine axlno- iCTHSferaae an d k - q c .dehydrogenase ac tiv ity m l iv e r and flUrtrgçnemiuB .muscle.The decarboxylation o f d(-oxo-lsocaproate i s analogous to the oonversion o f pyruvate to aoetyl CoA (M elster, 1965 • ) . The oofaotors required in the pyruvic decarboxylase reaction are TPP, Co ASH, Mg*+,36NAD* and lip o ic acid. McFarlane A Von Holt (1969 b ) investigated these oofactor requirements in l iv e r mitochondrial preparations and found that whilst NAD* or lipo ic acid or both did not sign ificantly influence the reaction, 2.0 mM Mg**, 0.1 mM Co A, 1.0 mM ATP and 0.2 mM TPP did. Sinoe I was using a crude homogenate, i t was necessary to re-investigate these requirements.The resu lts are presented in Pig. 1, showing that addition o f a l l or individual cofactors did not enhance dehydrogenase activ ity . One can only postulate that Mg**, CoA and ATP in the crude homogenate were present in su ffic ien t concentration to induce maximal ac tiv ity . Li polo acid is known to be bound firm ly to enzyme protein and w i ll not be removed during homogenization (Reed, 1960).Pyridoxal 5» phosphate ( Braunstein, 1960) and PAD (Beinert, 196O) are known to be oofactors fo r the aminotransferase. However, these oofaetors are a lso firm ly bound to the enzyme and the requirements were not Studied. < -oxo-g lu tarate is known to traJiM.imlnate read ily with L-leu cine (K eister, 1969 b ) and the substrate enhancement o f aminotransferase ac tiv ity by ¿-oxo-glutarate (» kg) was studied, together with the substrate saturation effeot o f L-leu cine. Fig. 2 shows that in l iv e r a t « kg 10 mM, l iv e r aminotransferase activity was s t i l l increasing with 15 mM leucine. As i t was possible to achieve saturation o f the enzymes with respeot toe kg concentration at 10 mM, an arb itrary deoieion was made fo r the assay medium to contain 10 mM L-leucine and 10 mM A-oxo- glutarate in Sorensen's phosphate buffer (25.0 mM) at pH 7»0. This concentration o f leucine was also chosen in the gastrocnemius assay because i t corresponded to the concentration fo r maximum activ ity found in muscle preparations and the increment in activ ity with concentrationsDOUCftCODiASE ACTIVITY (epaTIC. 1 . Determination o f co>factor requireaente fo r «KIC dehydrogenase a c t iv i ty i s l i v a r . F in al concentrations in 3.0 n ls -1 in 10 Horogenate (1 .0 « 1); leucin e 10 rii (0.9 ■ !); ok* 10 aK (0 .1 ml); Cofactora in Sorenasn'a b if fa r pH 7.0 ; ATP 1 .0 r ii ; CoA 0.1n.v. ; Co-carboxylaise 0.2 mH; Cytochrome C,0 .1 r ii and F«** 2 .0 r ii.DI2JME ACTIVITIES1— «¿DiC?KAKSFQiA£E5SAct&vltiaa (cp» 1l,C02/hr)a KIC dahydroganaaa a c t iv i ty . Tha assay conaiatad o f a Soranean's phosphata buffar (pH 7) 1 .0 «1; a 1 la 10 hoooganata 10 a * (1 .0 a l ) ; leucin a 10 BM (0.9 ■ !) and akg 1 0 aH (0.1 « 1 ) .t 'a Phosphate B uffa r.*.1of leucine above 10 nM was very small ( f i g . 2 ). However, i t was necessary to use 19 siM c ( kg to obtain maximal ac tiv ity in muscle.In it ia l experiments on boiled homogenate showed no catabolic ac tiv ity . However, in the crude homogenate, catabolism of L ( l14C ^ le u c in e was both linear with tiaw over a period of 60 min. and linear with respect to concentration o f the hosK>genate. This established that production o f '^CO^ and of O^KIC was the result o f ensyme activ ity .C ( i ) The E ffect o f Low-Protein feeding on Leucine Catabolic Ensymes in Liver and Gastrocnemius Muscle.McParlane A Von Holt (1969 b) suggested a block in the decarboxylnt ion oftftKIC as a resu lt of reduced 0 CKIC dehydrogenase a c t iv i t y in liver mitochondrial preparations from rats fed 20g protein/kg for 8 weeks. A sim ilar f a l l in the dehydrogenase a c t iv i t ie s for a ll three BCA's has also been demonstrated in liv er homogenates from animals given a d iet containing 90g protein/kg when compared with the response to a d iet containing SOOg protein/kg (Volhueter A Harper, 1970). Since oxidation of L [ l ,4C J leuc ine was reduced in rats fed a low-protein diet (Table 9) the effects of the d iet on ac tiv it ie s o f both the aminotransferase and dehydrogenase in liv e r and muscle were studied.C ( i t ) IH VITRO oxidation in C e ll-free extractsMeasurements were made with t [ l M c| leucine in tracer doses on tissues from rats maintained on the same dietary regimen as described in le c ti on I IA (i i ) . Only animals previously given the diet w ith an NDpiE ratio o f 0,10 were fasted. L irm r and**2gastrocnemius muscles were removed, frosen with solid CO^ and stored at -18°C for subsequent assay. Preliminaryexperiments revealed no deterioration in eniyse activ ities during the storage period. Leucine^C -oxo-glutarate aminotransferase E.C. 2 .6 .1.6. and 0t K1C—dehydrogenase activ ities were measured in both liver and muscle by the modified method used by McParlane A Von Holt (1969 b ) as described previously in Section I I B ( i i ) .C ( i i i ) RESULTSAssay o f the ac tiv ity o f the f ir s t two er.symes in the oxidative pathway o f leucine (Table 6 ) showed them to be present not only in l iv e r but a lso in the gastrocnemius muscle. Dehydrogenase ac tiv ity was c le a r ly demonstrated in the gastrocnemius muscle o f animals on a l l three dietary regimens, and was present in greater activity/mg protein than that found in liver.Muscle aminotransferase activity was even higher aad was approximately ten times as great per ng protein as that in liver. Thus the ra t io , aminotransferase activ ity : OC-oxo-acid dehydrogenase ac tiv ity was much higher in muscle. The enaymes in liver and muscle d iffe red not only in their levels of activity but also in their response to dietary stress. Low-protein feeding produced a r ise in aniscle aminotransferase ac tiv ity but a fa ll in dehydrogenase ac tiv ity to less than ha lf the control value. In contrast, liv e r aminotransferase and dehydrogenase activities were not changed appreciably. Thus in vivo conservation of injected L [ l , 4C)leucine (Table 5) was occurring without any change in l iv e r dehydrogenase activ ity , which hitherto has been held responsible for the reduced oxidation ofOC-oxo-isocaproicid in protein depleted unals (McParlane A Von Holt, 1969«Ab)Leucine K -oxo g lu tan te ami not rans ferase and g^-oxo iaocaproate dehydrogenase ac tiv it ie s of liv e r and muscle homogenates in rats either given a control or a lov protein diet or fasted for 48 hr. (Mean values with their standard errors for six rats per group)SECTION IlC(iii)TABLE 6d iffe r s ign ifican tly at the following le v e ls !- a, P < 0.01 | b, c, d, e and f , P<0.01.energy supplied by u tilis ab le protein l total metabolisableprotein.kHThe experiment!* with fasted rats indicated that changes in ensymc ac tiv ity (T ab le 6 ) could occur rapid ly, fo r within 48 hr, muscle dehydrogenase ac tiv ity had fa llen markedly.In contrast, l i v e r dehydrogenase a c t iv ity rose.D ( i ) The poss ib le r o le o f age in determining the response o f animals to p r o te in - f r e e feeding.The in it ia l experiments on leucine oxidation in vivo showed that 65g growing ra ts had the a b i li t y to reduce their oxidation o f leucine when growth was arrested on a low protein d iet (0.035 NH>:E). N eale (1971) observed that veanling ra ts given a protein-free d ie t were unable to conserve either leucine or valine when Lfu'^C }— leucine or L[u '*CJ—valine was given as a pulse dose in trag as tr ica l ly or intravenous ly . Purther studies (Neale, 1972) with «viscerated and control adult rats fed a protein-free d iet fa i le d to show any adaptation in oxidation by the peripheral t is su e s . Again the d i f f ic u lt ie s in the interpretation o f these experiments are dealt with in Section I V ( i i ) . Because o f these discrepancies, a further assessment was made of the a b i l i t y o f weanling rats to reduce the catabolism o f L fl J-leucine and valine on a p ro te in -free d iet, and the resu lts were compared with those in older animals. Furthermore, the experimental design included assessment o f liv e r and muscle ensyme ac tiv ities in r a t s at d ifferent stages o f developawnt.Thus, i t was possible to relate any changes in enzyme ac t iv ity to the a b ility to reduce the catabolism o f leucine and valin e.0 ( i i ) ANIMALS AND DIETSThree d ifferent groups o f female hooded L is te r rats were used to investigate the ox id ation o f leucine and valine and the<•5adaptation in the catabolic ensymos of these two branched- chain amino acids in animals o f different ages when given a protein -free (PP) d iet fo r 1( 2 or 3 weeks. The f ir s t group was obtained at weaning and immediately fed ad libitum a PP d iet . Two other groups of rats were fed in it ia lly on control d iet from weaning in which the ratio o f energy supplied by u t ilia ab le protein to tota l metabolisable energy (NDpiE) was 0 . 10 . When one group reached a weight o f approximately 85.Og the animals were given a PP diet for 1, 2 or 3 weeks.The last group was maintained on the 0.10 NDptE d iet until the rats had reached an average weight of 200g when they too were given the PP d iet fo r 1, 2 o r 3 weeks.Control animals received an adequate protein intake, i .e . 0.10 NDpiB, throughout and were assessed at weights o f 35, 85 and 200g body weights. A ll the aniaiala studied were fed until 5 hr before the experiment for reasons which have been discussed (Section I f A ( i i i ) ) . The rats were given an intragastric injection of a tracer dose o f L [l ,4C Jleucine or L (l ,4Cjvaline ( KjpCi/kg body weight in a solution o f 9g NaCl/1 with 10 ^ hnol leucine/kg body wt as c a rrie r - sim ila rly fo r valine) and placed immediately in a sealed vessel through which a ir was drawn at constant rate. The COj was trapped as described in Section I I A ( i i i ) . Oxidation during the f i r s t 3 hr. was expressed as the percentage of the labelled dose expired as ^CO^*D (1 i i ) PJZTME ASSAYSAminotransferase and dehydrogenase activ ities were measured in vitro in sniscle and liv e r preparations from rats given the same diets as those used for the assessment of oxidation in vivo.Groups of rats were k ille d by decapitation at weekly intervals•*6and Uieir liver* and gastrocnemius muscle* were excised and quickly frozen on solid C<>2. Pour enzyme activ ities were measured in both tissues < L - le u c in e -o x o -g lu ta ra te aminotransferase (pCrKIC) , L-val ine^t-oxo-glutarate aminotransferase E.C.2.6.1 . 6 . and oC -oxo-isovalerate dehydrogenase (*C- K1V) . Por the assay a weighed amount (500g) of liver or muscle tissue was homogenised by hand in ice-cold Sorensen's phosphate buffer (25 mmole/l) pH 7.0 to give a 1 HO (w/v) homogenate. Enzyme activ ities were determined by the methods described previously (Section I I B ( i i ) ) .D ( iv ) RESULTSTable 7 shows the proportion o f labelled leucine and valine excreted as CO2 by Jit three groups of an ia»ls . Veanling and 20Og animals excreted a sim ilar proportion o f label. Equivalent •mounts of *^C0^ were evolved from labelled leucine and valine in those animals given a normal diet and the individual values within groups were very consistent.The PP diet .ed to a decrease in oxidation rates in a l l three age groups. The response to a PP diet was rapid, since the greatest decrease in , 4C02 excretion occurred within the firs t week| thereafter further decreases in the catabolism o f either L ft ,4C )-leucine or , 4C ]-valin e were small (Table 7 ). There was a tendency for the youngest group of animals to have the greatest decrease in output, eg. there was a 59* decreasein leucine oxidation in the weanling rats compared with a decrease of 37* in the mature group. The adaptation in valine oxidation seemed less effective than that for leucine, with higher excretion rates o f , 4C02 from L [l ,4C Jval ine in rats on the protein-free diet.* 7In vivo oxidation of l £|14 c J leucine and L [ l 14C ] valine (1.0 ^*Ci/lOOg body vt) in rats on control (0.10 NDp:E+ ) and protein free (0.00 NDp:E*) diets. , 4C02 excretion in 3 hours expressed as a percentage of labelled dose given.SECTION IID(iv)TABLE 7Pigures in parenthesis indicate the number of observationsResults d i f fe r significantly (F < p .02 ) from the control value for the group.Ra'io of energy supplied by u t il is a b le protein to tota l sietabol isablep ro te in .*9nature o f the increase In most groups; frequently the highest leve l was seen a fte r 1 or 2 weeks and by the end o f 3 weeks aminotransferase leve ls were often almost the same as the in i t i a l control ac t iv it ie s . Thus, a fte r 1-2 leeks on a PF diet the ra t io , aminotrunsferae»:dehydrogenase activ ity in the homogenates had changed markedly, with an increase in the value from 2-3 i*1 thc control period to 5-22.tc t iv U t tJ la Mi . cX. fCa.trocnm.1.,..^.Measurements o f enzyme ac t iv it ie s in homogenates o f muscle (Table 9 ) confirmed the e a r lie r work (Table 6 ) that there was a higher aminotransferase:dehydrogenase ratio in this tissue than in liv e r . There were sim ilar changes < n the weanling and growing rats given the PF d ie t . Again, there was a de fin ite decrease in dehydrogenase a c t iv it ie s a fter 1 week, with further reduction by the end o f 3 weeks on the PF d ie t . In the oldest group o f animals there was a sign ificant decrease in ac tiv ity but, as In the l iv e r , the change was much less than in the younger animals. Thus, although the in i t ia l ac tiv ity in muscle o f X ETC dehydrogenase was much higher in the younger animals than in the mature (200 g ) ra ts , once adaptation had occurred, the younger animals showed a greater reduction in ac tiv ity than those in the oldest group. The aminotransferase ac tiv it ie s rose to high levels ■in the ea r ly phase o f feeding on a PF diet in a l l three groups but returned towards normal by the end o f the feeding periods.SECTION IID (ir )TABLE 8Liver ensyme ac tiv ities in ra ta given a protein-free (PP) diet»L— leucine and L-valine d -o x o g lu ta ra te hid i no transferase and K -oxo isocaproic acid (W^KIC) and K -oxoisovaleric acid (K-KIV) dehydrogenase ac tiv ities (u no In. amino acid or #t -oxo-ac id oxidised/ mg protein per h r .)(Mean values with their standard errors for groups o f four rats)LEUCINEMean Time on «C -oxog lut-Body PP diet a ra te aminoWeight (weeks) t ranaferase at- KIVWEANING 35gGROWING 85gADULT 200g8 .6 0.81 4 .9 9 .91 2 . 2* 0 .70 .4 9 9 .9 0 .70 .6 0 18.0* 9.90 .4 9 2 2 .9* 0 .91.09 0.8 0 .2 02 .8 0 .71 .9 0 .2 9 1.2 0.10 0 .4* 0 .097 . 012. 1*0.60 .90 .10 .29 .021.0*0 .9 9 0 .9 0 .9 0 0 .98 .9 0 .4 9 0.6 0.17 . 8 0 .61 0 .4 0 .96 . 6 0 .76 . 9 0 .1 90 .06 90 .0 60 .0 4 90 .044 .9 0.299.1 0.697 . 1* 0 .606 . 1* 0 .609 .9 0.990 .090 .9 90 .090 .10 .0790.0450 .04i d i f fe r significi(P.05)52Oxidation o f leucine in vivo was reduced in animals given a low protein d iet . Fasting, however, caused an increase in the output of 14C02 from L [1 ,4CJ leucine given in tragastr ica lly . Measurements of both leucine ot-oxoglutarate aminotransferase andpt-oxo-isocaproate dehydrogenase ac tiv it ie s in animals fed the control and low protein diets were carried out. Dehydrogenase ac tiv ity was demonstrated in skeletal muscle homogenates, although other workers had fa iled to show this (Volhueter A Harper, 1970). Furthermore, the low-protein diet led to a f a l l in muscle dehydrogenase ac tiv ity but increased liv er dehydrogenase ac tiv ity . Aminotransferase ac tiv ity in muscle rose in the low-protein ard fasted animals but the ac tiv ity in liv er vis unchanged.This work was carried out on weanling rats and contradicted the work o f Neale (1971). He observed that weanling rats given a protein- free d iet were unable to conserve either leucine or valine as judged by output o f 14C02< One reason for this difference may be that Neale used uniformly labelled amino acids. The second series o f experiments were designed to make fu rther assessments o f the ab ility o f weanling rats to reduce the catabolism o f I [ 1 14C ] - leucine and L [1 <4C ]-valine on a protein -free rather than a low-protein d iet , and the results were compared with those in o lder animals. Essentia lly the same results were obtained, showing that protein restriction in the diet led to a reduction in , 4C02 output from the radioactively labelled branched-chain amino acids.The results in Table 6 (Section H C ( i i i ) ) confirmed Young's (1970) observations that the greatest total B.CA. aminotransferase activ ity was located in skeletal muscle. Moreover, one can calculate the totalSUMMARY OF SECTION IIdehydrogenase capacity of both l iv e r and skeletal muscle from the datain Table 6 (Section I lC ( i i i ) ) and the reported tissue d istribu tion o f dehydrogenase ac tiv ity (Voihueter * H arper, 1970 ) . Liver accounts for only 2* of the body's dehydrogenase a c t iv i t y , with ^0.05?' fo r brain and {o.05?i for kidneyf their combined capacity is insu ffic ient to account fo r leucine oxidation in v ivo . I f muscle protein i s assumed to be 50J6 of total body protein, then muscle has the highest calculated total dehydrogenase activ ity . The sup position , therefore, is that muscle must play an important part in the regulation of branched-chain amino acid oxidation.Vith this as a working hypothesis, the second phase o f the work was begun. A hind-limb preparation was developed a'ong sim ilar lines to those described by Ruderman et a l (1971 ) and Jefferson et a l (1972), since this was essentially a more p h ys io lo g ic a l in vitro skeleta l muscle preparation than a homogenate. By constant infusion of labe lled amino acid into the perfusion fluid i t was p o s s ib le to deteimine quantitatively the oxidation o f leucine by skeletal m uscle in the hind- 1 tab preparation. Vith the same technique in vivo, as d esc ribed by Vaterlow A Stephen (1968) and later by Garlick A Marshall (1972), a quantitative estimate o f whole body leucine oxidation could also be obta ined . Muscle's contribution to total body oxidation of leucine could therefore be estiamted more accurately than had previously been done (Manchester, 1965). Moreover, the constant infusion method offered the opportunity to study protein turnover under conditions o f dietary s t r e s s both in vivo and in theperfusod hind-limb.5*LEPCITIK TUmJOVKR IN THE V7IOI,K BODY AND IT) Tl PHITOSKD HIND LIMBA. Estimation o f *4C02 retention in the bi carbonate pool.In order to measure the oxidation o f leucine, it is necessary f i r a t to know whether the ' 4C02 formed is quantitatively excreted.Recently i t has been shown that 20* o f an infused dose o f NaH14C03 was being retained in man (James, O arlick, Sender A Vaterlow, 1976) and that 80* was excreted as , 4C02. Therefore, i t was essentia l, i f accurate oxidation rates were to be measured, to estimate the retention, i f any, o f 14C0^. To do th ia , a tracer dose o f NaH,4C0j was infused constantly into the t a i l wain o f a rat and '^CO^ output monitored on the assumption that when I [ 1 i4C J leucine is decarbox- y lated , the , 4C02 gains d irec t access to the bi carbonate pool.Constant infusion of NaH14C(>3 in TiroFemale hooded rata weighing approximately 170g were fed ad libitum 0.10 NDpiE powdered d iet fo r one week and were then placed individually in g lass cyclinders as described in Section I I I B ( i i ) . The ta i l vein was cannulated (Section I I I B ( i i ) ) and NaH, 4C03 ( 3.0>Ci/ml in 0 .9 *sa lin e ) infused at a flow rate o f 0.48 a l/h r. CO^-fre* a ir was pumped over the animal (400 ml/min) and the efflu ent gases bubbled through three sequential hyamine hydroxide/ethanol traps. production was monitored fo r 3 hours to estimate the amount of labelled , 4COa retained within the rat tissues and the h a l f - l i f e of the bicarbonate pool.RESULTSFig. 9 shows that , 4C02 output reached plateau spec ific radioactivitySECTION IIIC02 output (dpm X 103/^in).(dpa * 105/^ln).Fif l 5r.g jOutput of <*UT’r'S constant infusion by ta il vain of N a H ^ C O j (3J0 ||Ci / ml in 0.9% salina)into rttraa rats (180 g body w t.)within 60 minute« and that the calculated h a l f - l i f e o f thebicarbonate pool ia 15 minutes. This is in agreement with the estimate made by Millward (1970) of 12$ minutes. Furthermore, the figure shows that there is neg lig ib le retention o f inthe bicarbonate pool unlike the findings o f James, Garlick, Sender apd Vaterlow in man, where 20?! was retained. Therefore, no correction was necessary in estimating absolute oxidation rates in subsequent studies involving the constant infusion o f L [t M C ) -leuc ine, on the assumption that the , 4C02 decarboxyl ated immediately enters the bicarbonate pool.B ( i ) LEUCINE TmVOVKK IX THK VHOIJC RAT IN’ VIVOE arlier experiments both in this laboratory (Section I I ) *nd in others (McFarlane * Von Holt,t969a| Neale 1971, 1972) have estimated the rate of B.CA oxidation with an in tragastr ic , intra- peritoneal or intravenous pulse dose o f radioactive tracer amino acid . Unless large quantities o f cold amino acid are added the precursor »p a c i f ic rad i oac t i vi ty in the various tissues w ill be changing rapidly. Therefore, the constant intravenous infusion technique described by Garlick k Marshall (1972) was e«q»loyed to measure more accurately the rates o f leucine oxidation in vivo.Vith infusion o f a constant tracer dose o f L [ ) 14C ] -leuc ine the precursor pools for oxidation and protein synthesia ( in tra-ce llu lsr pools) reached plateau spec ific radioactivity within 2 hrs (F ig .« ), and the level was maintained to the end o f the in fusion . Similar results have been obtained with the infusion o f a number of d iffered amino acids (Oan h Jeffrey, 1967| Vaterlow A Stephen, 1968» Garlick h Marshall, 1972| Seta et ml, 1973). In liv e r the plateau SR of free leucine reached approximately 30% o f ttmt o f plasma whilst in57the gastrocnemius muscle i t vaa in excess of 70^ ( f i g . 8 ) .The fact that the specific rad ioactiv ity o f the free leucine at plateau was d iffe ren t in the plasma and the various tissuesas indicative o f e ffective compartmentati on. One reason fo r the lower specific radioactiv ity in the tissue is dilution o f the in trace llu la r pool by unlabelied amino acids (leucine) derived from proteolysis (Gan ft Jeffuy, I967| Waterlow ft Stephen, 1968).) METHODS(a ) Rates of oxidationIn order to determine rates o f leucine oxidation the fo llow ing experimental protocol was used. One group o f female hooded rats (Anistal Suppliers (London) Ltd) weighing approxiamtely 170g were fed ad libitum fo r one week on a powdered d iet (Net dietary protein i energy rat io , 0.10 NDptE) which maintains normal growth rates. A group of rats was then infused via the ta i l vein with a tracer dose of L f1 14C ]-leuc ine (S.oyiCi/ml in 0.9% • a lin e ) continuously fo r 6 hours (0 .4 8 ml / h r ) . Another group was fed a protein- free powdered d iet fo r one week and then infused in a sim ilar manner. The protein—free d iet was iso - energetic with the O.lONDpiB d iet (see Table 4 ).The an isw ls were 'tra ined ' fo r 2 days before the infusion o f labe lled amino acid to s it quietly in a g lass cylinder, stoppered at e ith e r end, but ventilated adequately (see f i g . 6 ) . This procedure acclimatised the rats su ffic ie n t ly fo r them to s i t happily throughout the 6 hour infusion periods while '^CO^ output was being aionitored. The ta il o f the rat was passed through a small hole in one o f the rubber stoppers and fixed in place with adhesive tape to prevent i t bring retracted into the tube by theFIG 6* AFfAHATUS FOI THt C O U K flO N O f C O , DUUNG INFUSIONS »1 VIVO59rat when the ta il vein was cunnulated (method described in Section I I I B ( i i ) ) . CO^—free a ir was pumped through the cylinder at a rate o f 400 ml/min and then through three sequential traps of hyamine /ethanol mixture with phenolphthalein indicator. Immole CO^ was considered to have been trapped when the indicator changed from pink to colourless as i t had then reacted with 1 m mole of hyamine hydroxide in the f i r s t v ia l.The time taken to trap 1 ■ mole of CO^ was recorded throughout the experiment. Thus both the total amount of CO ^ produced/hr and the specific radioactivity of the CO^ could be detenained.ICtail toluene t PPO ( 2 , 5 dinitrophenyloxasole, 4g/l toluene) was then added to the v ia l and immediately counted. The efficiency of counting was 7Apparatus for **co2 collection from L f l14C)-leucine infusion A ir was pumped at a rate o f 400 nl/nin through CaCl^ to remove water vapour and thence to sequential 20ml volumes o f saturated Ba(0H)2 in Quickfit bubble traps (MF 24/3/S) to remove C<>2. The C02 free a ir then passed again over CaCl2 into the g la s s cylindrical chamber ( f i g . 6 ) . The cylinder (25cm x 6cm I.D ) was stoppered at either end with Oallenkamp No.57 rubber bungs. The in le t bung had 2 bore holes (8mm. I.D ), one fo r the passage of incoming CO2~free a ir and the other fo r the animal's ta il to pass through.The outlet bung had one 8mm bore hole which led by the connecting Tygon tubing ( 3mm O.D x 1.5mm. I.D ) to a further water vapour trap and thence to a v ia l containing 3 ml o f a 2i1 hyamine hydroxideiethanol mixture with phenolphthalein as ind icator.Two more vapour traps and v ia ls were connected in se r ie s to the f i r s t trap to check C02 trapping efficiency. Trapping in the60f i r s t vial was about 98^ effic ien t. Preliminary work involving the constant infusion o f MaH^CO^ showed that there were no leaks in the system, especially where the ta il passed through the bungs (Section IIIA ).Rates o f production were calculated as dpm CO ^ evolvedper minute of infusion. By constantly monitoring the output of throughout a six hour period, the rate of rise to plateau specific radioactivity could be measured. From these data it was possible to estimate not only total body leucine flux but also the flux through the oxidative pathway.(b ) Rates o f protein synthesisFemale hooded rats (Animal Suppliers (London) Ltd) were obtained at 170g and fed ad libitum a powdered d iet (0.10 NDpiE) fo r one week. Another group of sim ilar rats were fed a protein—free powdered d iet (0.00 N’DptE) for one week. Animals from both groups were then infused via the ta i l vein with a tracer dose o f leucine in the manner described below. Aniaal* wereinfused for 2 or 4 hours at which times they were sacrificed by decapitation. Nixed venous blood was rapidly spun and a known volume of plasma added to 2ml o f cold 3.00 sulpho -aalic y l ic acid (S .S .A ) to precipitate protein. Semples o f both liv e r and gastrocnemius muscle were also rapidly taken, homogenised in cold SSA and stored at -18°c. Measurements o f free and bound leucine specific radioactivities were carried out on a Locarte amino acid analyser fitted with a column effluent stream sp litte r (Fern A Oarlick, 1973). Fractions were counted in 10ml o f 0 .4 *PPO (2,3 dinitro phenyloxasole in a mixture of toluene:triton X-IOO (2 :1 ) at an efficiency of 83-90*. Results for free and61bound leucine specific radioactivity a t 6 hour« were obtained from tissues removed from animals used to determine oxidation rates.Tail Vein Infusion of Labelled Amino Acid A 20 gauge disposable hypodermic needle was separated by dissolving its fitt in gs in chloroform and inserted into a length o f narrow bore polythene tubing (0 .4 mm I.D ). The other end o f the tubing was fixed to a hypodermic syringe containing physiological saline. The ra t was then wrapped in a hand-towel to restric t it s movement du ring the infusion period. Its t a i l was le ft free to be in se rted into warm water for a few minutes both to clean i t and to increase the blood flow. The t a i l was then cleansed with x y lo to l which also makes the vessels d ila te . The needle was in serted into a la tera l vein and firm ly held in place with adhesive tape. The needle and tubing were cleared of blood by in je c t in g approximately O.la l of sa line . The cannula was then attached to the syringe portion of a continuous infusion pump. A so lu tion of L [t ’ V J loucino in physiological saline was infused a t a rate of 0.4Sml/hr (i.tyiCi/sl prepared by dissolving solid L [ l J-leucino of spec ific radioactivity 6omCi/na»l in saline without any ca rrie r).• ( i i i ) LKUCIKE OXIDATION RATES IV VIVOCAUSATION - I f one assumes that fo llow ing decarboxylation o f ** (1 J-leucine »•»*»» direct access to the bicarbonate poolthen the oxidation rate for leucine in the whole rat is given by the following equation when plateau s p e c i f ic radioactivity o f , 4C02 output has been reached.62SR, 4C02 at plateau (dpm/min)E ” SR frea leucine in muscle ICP ( dpm/^ lmo1e )This ca lcu lation ia baaed on the aaaumption that oxidation occurs in a pool in which the SR o f froe leucine is equal *r sim ilar to that is muscle. It gives rates as ^imoles/sin but in Table 10 the oxidation rates have been expressed as ^baoles/hr/lSOg rat in order to obtain a better comparison between control and rata fed a p ro te in -free d ie t .RESULTSP ig .7 shows the r ise to plateau in spec ific rad ioac tiv itywhich ia attained in l i t t l e over 2 hrs in the w e ll- fe d group and after a l i t t l e longer in the p ro te in -free group. The spec ific radioactivity o f free leucine in the gastrocnemius muscle (Table 10) was taken as a represents*‘ re average of the precursor specific rad ioactiv ity o f a l l tissues in which oxidation of leucine occurs. Since the SR in the precursor pool (GASTROCNEMIUS I CP) . »d ia the pool from which 14C£>2 » • derived (HCOj—) had reached plateau by 2 hours and since plateau was maintained for up to 6 hours ( f i g . 8 ) i t was possib le to estimate the rate o f leucine oxidation from these two values. Because o f the difference in mean body weight for the two groups, figures were adjusted so that results were expressed p>r 1 SOg body weight.As can be seen from Table 10, the output o f *4C02 (dpm/min) at plateau spec ific radioactiv ity in the w e ll-fed group was greater than in the group fed the protein free d iet . When rates o f oxidation are determined, with adjustsMnta for the spec ific radioactivity o f the gastrocnemiua muscle ICP pool, then there ia a reduction from 29.90 ¿w»>le*/hr/lBOg body wt. in the well fed group to 20.50 Jbnol*a/hr/l80g body wt. in the pro te in -free group (Table lO ).TABLE 10ABSOLUTE OPDATIOM RATES OF L|1U C 1 leucine IN VIVO (»mole» 'hr/l80g rat) E ffoct o f protoin -froo feedingFigures gieen »rt Means - 8.S.D iffer» s ig n if ic a n tly from con trol value p ^0.01s n « 7Output -where Sp - the SR o f plasma lysine at any time t|SpM T * the plateau SR o f plasma lysine \ p i s a rate constantX p fo r ly sin e was shown to be from 12-24 days-166(Vaterlov A Stephen, 1967) and fo r tyrosine 00 days” (Garlick et a l , 1973)» In the present work, Ap fo r leucine was found to be 33 days-1 .S im ilarly , the time-course o f SR o f the free amino acid in thetissue (S i ) can, without sign ificant erro r, be expressed as a s in g le exponential i Si = Simax (1 -e ” '^l t ) eq u .(3 )The problem is to determine the value o f A i . This question has been discussed in de ta il by Garlick et a l , 1973)* From th e ir conclusions i t was considered that under the conditionso f the present experiments i t would be appropriate to take 1 m A p “ 33 days” 1 This approximation is Justified when the z a t io o f protein bound to fre e amino acid is la rg e , as i s the case with leucine in muscle and liv e r . Substituting eqn. (3 ) into eqn. ( l ) , taking A l - Ap, and integrating, gives ij s ■ A esi A P -K .Q -.-V ) •qu .(4 )A p ■- an equation o rig in a lly derived by Swlck (1930). This equation can be solved graphically fo r and enables the rate o f protein synthesis in l iv e r and muscle to be determined from the experimentally measured SRs o f leucine in the protein and free amino acid pool o f the tissue at the end o f the infusion . Accurate determination o f i s unnecessary, since la rge variation in i t s value resu lt in only small variations in (Waterlow A Stephen,1960). K( , the fraction a l rate o f synthesis, i s expressed as the% o f protein mass replaced each day.67v) RESULTS(a ) Effect of protein-free feeding on muscle synthesis rates Plateau SR o f free leucine was attained w ith in 2 hours of starting the infusion in both plasma and gaatrocnemius muscle ICP ( f ig .S ) . The plateau value was maintained over a 6 hr period (Table 11) ana incorporation into sk e le ta l muscle protein was linear. Peeding a p.'-ta in -fee d iet led to an increase in plasma SR of free leucine. This is consistent with observations made by Garlick, M ill ward. James * Vaterlow (1979 ). At the same time protein-synthesis in the gastrocnemius muscle was reduced from 9.3* D ' to 6 .0 * D The synthesis ra te s observed in gastrocnemius were sim ilar to rates o f p ro te in synthesis estimated in the same muscle o f lOOg Vi star rats but in fused with U,4C- tyrosine (Garlick at a l , 1973).(b ) Liver synthesis rates in vivoAs with the 3R of miscle ICF, the specific rad ioac tiv ity of intracellu lar free leucine in liver reached a p lateau by 2 hours which was maintained up to the end of the 6 hour infusion ( f ig .S ) . Peeding a Of NDptE d iet led to a small but s ig n if ic a n t rise in the plateau SR ia liv e r ICP. This is consistent with data of Oarlick et a l (1973). Protein synthesis was increased from 50*D-1 to 121.0* D-1 on feeding the prote in -free d ie t fo r 7 days (Table 12). Oarlick et a l (1975) found that l i v e r protein synthesis rates increased in 10Og Vistar rats from approximately 50* D 1 to 94* D * by the 9th day of protein— fr e e feeding. Their results were obtained by the constant infusion o f U. ,4C tyrosine.« • «Specific Radioactivity (SR) of 1(1 ^ Q -le u c in e in plasma, liver ICF a id gastrocnemius muscle ICF in rats (« 1 » g ) fed 10% NDp;E a 0% NDp:E diet f a 1 week10% NOpcK 0% NDp*0 plasma^ **Cl£ T t O H H STMTHKSIS RATES IM V1TO Spacific rndioactlvity of laue in* in pUsma,gkitroenamu* ICF and auclt fntoii in rata fad aithar 10* NDp:B or 0* NDp:E diat for ona «**k and than infuaad. Practional aynthaaia rata* of nuacla protain ar# calculatad aa daacribad in 1b* Wat. lata «ara infuaad with a tracar dosa of L[l14c}l#ucin* for2, 4 and 6 kr nt a rat* of 0.4« nl/hr (9.0 pCi/ml in 0.9< salina)UUCIVE SPECIFIC RADIQACTIVI TT (dpn/nmolc laue in*)/-- 1--------- \TIME D iatary Group Plasma Gaatroc ICP QaatrocProtaia Muacla Protain(houra) SDp:E * (9p) (S i) (S_) Syntheaia Rat*’ ( S o - ')2 ) 43.4 - 2.6 32.7 * 2.1 0.192 - 0.079 10.00 )4 ) 10 90.2 - 9.4 30.9 • 2.9 0.371 - 0.140 8.81 ) 9.32 - 0.61* ) 40.4 - 7.4 29.3 - 3.6 0.980 - 0.100 9.19 )2 )i61.7 - 13.6 30.9 - 2.4 0.120 - 0.048 6.69 )4 ) 0 77.2 - 9.9 31.« - 2.9 0.249 - 0.093) # A.9.74 ) 9.97 - 0.640 ) 63.7 - 10.3 32.1 - 3.7 0.389 - 0.090 5.48 )Maans - S.D.Di ffara aignificantly fron control nuacla protain ayntbaaia rat* p3 showed that there was neglig ible retention of ' 4COj in the bicarbonate pool.The data on rates of flux and oxidation obtained in t h is way are presented in Table 13.These results can be converted to ratstof protein t u « " o » » r i f it is assumed that 334 /»moles leucine are contained in 1g protein (Pern, 1975 - PhD Thesis), Furthermore, i f 20* o f body veight is protein then the fractional synthesis rate o f protein in the whole body is approximately 18* D~' in the well fe d 180g rat. This rate agrees quite well with that obtained by Gmrlick et a l (1973) of 40g/kg body wt/day estimated by the in fu s ion of K,4C tyrosine. James (1972) reported similar resu lts w ith the infusion o f both ,4C lysine and ,4C glycine.B (v ii ) DISCUSSIONI f we consider the estimates derived from measurements on plasma (Table 13A), then on changing from a 10* NDpiE intake to m O* wDp jE diet leads not only to a reduced total body flux bu t also to a reduction in total body synthesis and oxidation o f loucine. The breakdown rate shows very l i t t le variation. O'Keefe o t a l (1974) also observed that in patients who had undergone e lec tiv e surgery, synthesis rates and oxidation rates determined in this manner decreased, but breakdown rates were unaltered. Thoao75measurements were based on plasma plateau values and on theassumption that "free leucine within tissues equilibrates with plasma rapid ly enough to form a s in gle frec leucine pool".The results in Table 13 show that these assumptions have to be questioned. When the plateau SR o f free leucine in gastrocnemius is taken as representative o f that o f free leucine in the whole body, then the interpretation of the resu lts ia en tire ly different. Oi feeding a protein -free d ie t (Table 13B) the tota l flu* does not a l te r , nor does the synthesis rate. The oxidation rate is decreased while the breakdown rate is increased (Table 13B).What is the reason fo r this discrepancy between the two methods • f estimating flux and the values derived from it? The discrepancy arises from the fact that the de fic ien t d iet changes the relationship between the specific rad ioac tiv ities in plasma and muscle. The plateau SR's are shown in Table 14.and hence the and hence theOn the p rote in -free diet the plasma SR is h flux is less. However, the muscle SR is th. plateau SR in muscle (SRm)r* tio p U t i ^ S R " l i - 5Tf^ i7 (SR ^) 11 r*duc#d from 70* in the rats on the normal d iet to about 50* on the protein free d iet. This means that there is more internal recycling o f amino acid. The d#dre# ° f recycling, R| i .e . the proportion o f amino acid derived t rom protein breakdown which is taken up again into protein within the c e ll is given by the relationship derived by Aub A Waterlov (!970)iR - ,-SRm' SHpTherefore, the conclusions to be drawn from Table 14 seen to be that on the protein free diet the 'plassw ' flu x , representing the exchange o f amino acid between tissues, is reduced, but77recycling within the tissues is increased. How these changes aro brought about needs further work. Moreover, the conclusion can only be tentative, because i t may not be correct to take the SR in gastrocnemius as representative of the whole body.C ( i ) LEUCINE TURNOVER IN THE PERFUSED HIN'D-LIMBThe hind-limb perfusion technique used in the present work w ill be described in detail as it was a modification of the methods described by Ruderman et a l (1971) and Jefferson et a l (1972). Preliminary measurements showed chat 80^ by weight o f the hind- limb was attributable to skeletal muscle and connective tissue, 19^ to skin and ta il and the remaining 5jt to bone. As the major vessels to the skin were tied and the ta il constricted by a tight ligature, the preparati«,,. was essentially a ske leta l muscle perfusion.By infusion L£l 14C ] leucine and measuring ,4C02 output and the SRs of free leucine in the in tracellu la r free pool o f gastrocnemius and in protein i t was possible to estimate rates o f both leucine oxidation and protein synthesis. Thus the contribution o f skeletal muscle to total body leucine oxidation and protein synthesis could be determined.C ( i i ) THE PERFUSED HIND-LIMB PREPARATIONThe operative procedure involved ligation of superficia l vessels to the abdominal wall and akin o f the hemicorpus followed by pelvic evisceration, ligation of major branches o f the great vessels and fin a lly , cannulation o f the aorta. The liv e r was then excised at the level of the posterior vena cava and the blood allowed to drain free from the transected hemicorpus.78Details o f tho operation wert aa fo llow s:-Rats ware anaesthetised by intraperitoneal injection ofphenobarbitonC(Pularin « lOO U/lOOg body wt).The baae of the ra t 's t a i l was then Hgated and a aid -lin e incision made in the abdominal wall from the pubic symphysis to the xyphoid process. The incision was extended la te ra lly towards the kidneys. The superficial ep igastric arteries ( l ) to the abdominal wall together with the saphenous branch (2) and the superficial vessels to the skin (3 ) were ligated on both sides ( f i g . 10). A fter this the in ferior mesenteric artery (4 ) and part of the descending colon (5 ) were ligated. The colon was pulled forwardi the ovarian (6 ), uterine, pubic epigastric trunks, bladder and uterus (7 ) were ligated and the whole reproductive tract and bladder dissected out.Next, ligatures were placed round the suprarenal and renal vessels (8 ) and the kidneys removed. The coeliac and superior SMsenteric vessels (9 ) were also ligated. An incision was then made into the thorax and a loose ligature placed round the descending aorta above the level of the diaphragm. The aorta was clamped above the looae ligature and an incision made in its wall, into which the canula (blunt ended needle « serum sine '0 ') was inserted and tied firmly in p lace. The perfusion was begun (10 nl/min) and the Spencer Nells clamp was rearaved immediately. The liv e r was quickly excised above the posterior vena cava and the animal transected above the level o f the aortic cannula. The fa t pad a overlying the psoas muscles were then carefully diaaected out. The perfusate was allowed to flush the residual rat blood from the hemicorpus for a period Of 4-9 minutes. The preparation was then transferred to a stainless79F IG 10H IN D LIMB PREPARATIONLigatures of the rat hind-limb preparation (far details see text)80i t « ( l perfusion box and the perfusate recycled. The entire operative procedure lasted approximately 8 -10 minutes.Perfusion ApparatusA schematic flow chart o f the 'Perfusion Apparatus' is set out in f i f .1 1 . Essentially the apparatus is sim ilar in design to that described by Ruderman, Houghton A Hems (1971), consisting o f two pumps (Vatson-Marlow H.R. flow inducers), a convoluted glass oxygen chamber, reservo ir, sphygmo-manoraeter and animal trough. They were a l l connected with tygon tubing. Sterilised 'Poplin ' cotton material was used in the f i l t e r .The perfusate reservoir had an approximate capacity of 50ml and in the bottom was placed a small magnet. This was rotated during recycling o f the perfusate by the magnetic s t ir r e r placed under the reservo ir. The reservo ir had three outlets. Prom one, the perfusate was drawn by pump No.1 and pumped v ia the f i lt e r to the top o f the convoluted g lass oxygen chamber over «.-hose surface the perfusate filmed. Humidified i CO^ (95/9) flowed counter- current to the perfusate at a Known fixed rate. It was possible to co llect the gas leaving the top of the chamber in order to estimate The perfusate pooling at the bottom of thechamber was able to overflow back to the reservo ir by the 2nd reservoir opening or it could be pumped (No.2 ) v ia the sphygmomanometer (to measure in line pressure o f the perfusate) to the hind-limb in a closed stainless steel animal trough.The perfusate flow rate (lOml/min) into the hind-limb was kept constant by pump No.2.The animal trough was constructed o f stain less s te e l (length 17.9cm| width 10cm| depth 9 . 2cm) with a central d u ll angleddepression fo r easy co llection o f perfusate drain ing from the82vena cava of the perfused hemicorpus. The outlet from the end vail o f the trough le d back to the third opening o f tha reservo ir. The can 10; KH^C*, lO. This mixture is gased with 95* i 9* C02 fer 19 minutes at 0° to 4°C prior to adding the NaHC0} (90 ml). This83lowers the pH and prevents precipitation o f calcium bicarbonate* The buffer is gassed for a further 10 ein. 1Hmg/l tyrosine and 0.3*1 (stock) I M pyruvate were added before the perfusate was Bade up. Tyrosine was added at this stage because owing to its In so lu b ility i t is not posrible to prepare i t in a stock solution o f essential amino acids.Human aged blood (21 days o ld ) was centrifuged at 2300 rpm for 15 min at 4°C in an MSE centrifuge and both plasma and leucocytes aspirated by pasteur pipette from the top of the erythrocytes.The erythrocytes were then washed three times with the Krebs- Henseleit bicarbonate bu ffer, each time the supernatant having been removed after centrifugation.The perfusate was then prepared from the ««ashed erythrocytes (90 m l), bovine serum albumin (60 ml o f 15* solutation, w/v), non-essential amino acids (0.8 ml), insulin (0.4 ml stock) and glucose (1.0 ml of 20* solution, w/v) together with heparin (0.2 ml o f 1000 U/ml solu tion ). The volume was made up to 200 ml with Krebs-Henseleit bicarbonate buffer so that the final concentration o f albumin was 4.5*1 glucose 5.55 aH and o f amino acid as in Table 15.Stock solutions required for preparation o f this complete medium were as fo llow si-(1 ) A mixed solution o f essential amino acids' details are liated in Table 15.(2 ) A mixed solution of non-essential amino acidai details im Table 15.(3 ) 20* glucose solution ' 20.Og D-glucose was dissolved n bicarbonate buffer and made up to 10(fel.8<*CONCENTRATI ON OP AMINO ACID MIXTURES FOR HIND-LIMB PERFUSIONS• ) Non-Essential Aminio Acid Mixtiurs - 2 x PlasmaMol.Vt. Cone, in Stock Solution (M)Cone, in Perfusats 1 Amino ARGININE ■ISTIDINE ISOLATICI NK LEUCINE LISINE METHIONINE HBNTLALAN INE THREONINE TRIPTOPHAN TIROSINEid Mixture174 0.100209 0.050151 0.050131 0.050146 0.175149 0.050165 0.050119 0.125204 0.050117 0.0100.200.100.100.100.350.100.100.250.10(0 .10)0.20VALINE(4 ) 250 BiUnit/ml insulin solution.(5 ) Borine serum albumin solution; 66.Og o f bovine serumalbumin (Pentex Praction V, Rei•search Division. MilesLaboratories Ltd ) was dissolved in 240 ml bicarbonatebuffer. When the albumin was dissolved 13.2 ml ofN-NaOH (1 ml o f N-NaOH to 5« of Albumin) was added toitra lise residual fatty acids. This solution wasdialysed against three changes o f bicarbonate buffer for 1 to 2 days using Visking tubing (Ga)lenkamp 36 x 32 mm). Dialysed albumin solution was diluted with bicarbonate bu ffe r to 440 ml to give a fin a l concentration o f 15g* (w/v).C ( i i i ) VIABILITY OP THE HIND-LIMB PRKPAkATlONSome o f the most sensitive indicators o f the v ia b ility of the perfused hind-limb are potassium efflux , ATP and creatine phosphate concentration in skeletal muscle, tissue water content, oxygen upteJte and visual appearance (Rudeman (Thesis) 1972)| Jefferson et a l (1972). In a control perfused hind-limb,minute perfusion was almost the same as at the start. Insulininto red c e lls since th is uptake was not observed in recycled perfusate alone.However, when the perfusion period was extended to 125 minutes. Ruder man found that perfusate potassium tended to rise a fter the f i r s t 65 minutes. He suggested that this may have been due to red ce ll haemolysis which occurs in a l l perfusions, and a f a l l in perfusate pH due to lactate accumulation. RudermanRuderman showed that the mean K* level at the end of the 35(12.5 mV/mi) caused a net uptake of I + which tabi.a fte r 5 minutes. This not due to87PROPERTIES OP THE PERFUSED HIND-LIMB PREPARATI ONCRITERIA IN VIVORudennan et a l (1971)Jeffersonet al (1972) present workF .r fu .i™ <■*"> -120 180120Water Content (»1/100«)(1 ) Psoas(2) Gastrocnemius74.0 - 0.6 73.4 * 0.6 _74.5 - 0.5 74 .« - 0.473.6 - 0.4 73.0 - 0.56.02 t 0.19 5.2 * 0.3 6.14 - 0.15 -(junoles/g)Creatine phosphate 15.04 - 0.29 15.10 - 0.60 14.58 - 0.58(psoles/g) Insul in (-U /-D 0.0512.5 25.00.25k* 0-1^0\mEq|lj -1.0Perfused Flowrate 12.0 10.0< »l/ »in )Perfusion pressure 137 - 4 9 0 - 1 0<-»«*> ------------------------ -Protein Synthesis rate in Gastrocnemiu«Oft»"’ )(1 ) 1<* NDptE diet 9.32 - 0.61_ 8.85 - 2.35 4.69 - 2.06(2 ) O* NDp:E diet 5.97 - 0.64— .Protein synthesis rates in the hind-limb also found tobe comparable to rates o f skeletal muscle protein synthesis in the whole animal. This parameter is certainly a good indicator o f limb v iab ility . For rapidity o f determining the v ia b ility o f a limb, the continuous monitoring of (released during the catabolism o f L [l ,4C Jl eucine )vas frequently used. In it ia l studies suggested that the preparation was viable for 2-3 hours but with experience gained one could only guarantee a viable preparation for at least 2 hours. A fter this period of time, the pressure on the perfusate input side of the hind-limb began to rise slowly, thus interfering with the oncotic pressure between plasma and muscle ce lls and causing oedema. Haemolysis and hence I leakage became a problem and the pH of the perfusate would begin to fa l l from pH 7.4 and approach pH 7.1. It was therefore decided that the longest experimental preparations should onlv la st 2 hours. This proved to be a disadvantage as plateau specific radioactivity of 14C<>2 output had not been obtained in 2 hours ( f i g . 12). Work could obviously be carried out to increase the length o f tine for which the preparstion ia v iab le , thus giving greater accuracy in estimating absolute oxidation ratea. Vith experience it was possible to re ject those preparations which would not have survived a 2 hour perfusion. The visual appearance would immediately indicate any onset o f anoxia or oedema and the arteria l pressure was a sensitive indicator o f v iab ility , as was the 14C02 output, which could be measured immediately.Although not n il the parameters which have been used as acheck by other workers were examined, I believe, with a certain degree o f confidence, that the present resu lts are based on preparations as v iable as those reported in the lite ra tu re . The finding o f synthesis rates in the hind-limb preparation comparable to those in the whole animal adds c re d ib il ity to this assumption (Table 16).C ( iv ) METHODSPemale hooded rats (Animal Suppliers (London) Ltd) weighing approximately I70g were fed ad libitum for one week on a powdered d iet (0.10 hDp:E) which maintains normal growth rates. A group o f rats was then used for the hind-limb preparation as described previously (Section I I I C ( i i ) ) .Once recycling o f the perfusate had cossnenced, a tracer dose o f L [l ,4C Jleucine (5.0JICi/ml) was constantly infused at a rate o f 0.48 ml/hr into the reservoir ( f ig . 11 ) . The constituents o f the perfusate were sim ilar to those described previously (Section I I I C ( i i ) except that the amino acid concentrations were increased above those norsmlly found in plasma in order to compensate fo r amounts removed by net protein synthesis and oxidation. The amino acid composition of the perfusate was also varied according to the previous dietary state o f the animal. Normal plasma concentrations of amino acids in the rat were taken from the resu lts obtained in this laboratory (Table l ) . Amino acids were added to the perfusion medium to give the following concentrations in terms o f normal rat plasma ( l ) Bata on a normal d iet i non essential amino acids 2.5 x plasma, essential amino acid, 2.0 x plasma(lOjC NDpiE, HiRh a 'a ) . (2 ) Rata obtained at I70g and fedad libitum the protein-free d iet fo r one week; non eaaential amino acids 3 .5 x plasma, essential amino acids 0.75 x plasma (OjC NDp.E, Low a ' a ) .Insulin was added to the aedium (250 ^lU/ml) only in the preparations f r o a rats on the norautl diet (lO^ NDp:E). These concentrations o f amino acids were chosen in order to maintain both protein syn thesis rates and amino acid concentrations at sim ilar lewels to those found in riwo. Preparations o f hind- limbs from an ism ls fed the normal and protein free d iets were also perfused in an identical manner but the concentrations of amino ac ids in the medium were exchanged between the groups to determine the possible role of amino acid concentration in the regulation o f leucine oxidation and/or protein synthesis. The four groups perfused were thus designated : - GROUPf tOjt NDpiM, High a 'a .2 10jt m*>«E, Low a 'a .) Oft NDp :E, High a 'a .4 O* NDp «E, Low a 'a .Insulin was a ls o infused (70 ¿iU/hr) into a l l groups o f hind- limbs to compensate fo r losses due to adhesion of the insu lin to the g lass w a l l o f the lung and reservoir and possible degradation by the perfused hind-limb. O^/CO ^ (95*5) was used to gas the perfusate at 300 ml/min and was then bubbled through 3 ml o f • 2 i I mixture of hyamine hydroxide/ethanol with phenolphthmlein was an indicator (Kaihara I Vagner, 1968). Immediately the hyasine had been neutralised a new r ia l was91substituted. A second bubble trsp - in series with the f ir s t - showed that trapping of ,4C(>2 in the f i r s t v ia l was 98* e ffic ien t . The time taken to trap 1 nmole of CO^ (1 MOLE Hyamine reacts with 1 MOLE o f C02> was recorded throughout the experiment. The SR o f the C02 could thus be determined. lO ml toluene s PPO (2 , 5 dinitrophenyloxaaole, 4.0g/l toluene) was added to the v ia ls which were counted in a Beckman Liquid Scin tilla tion counter (Model LS-150). With the external standard ratio method, efficiency o f counting was 75*. Samples o f plasma were taken at regular intervals| at the end of a 2 hour perfusion plasma and gastrocnemius samples were taken rapidly fo r estimation o f SR of free and protein bound leucine by ion-exchange chromatography on a Locarte amino «r id analyser fitted with a column effluent stream sp litte r (Fern ft Carlick, 1973). Fractions were counted in 10 ml of 0 .4 * iTO in a mixture of toluene i triton - X-100 (2 « l ) at an efficiency of 85 - 90*.v ) Oxidation o f ?euclne in the M n d -ll»b .Rates of ,4C02 production were calculated as dpm 14C<>2 evolved per minute o f infusion time. In order to estimate the absolute rate of oxidation o f leucine by the hind-limb and the rate of synthesis of skeletal muscle, the SR of leucine in the precursor pool was taken to be that o f free leucine in the gastrocnemius muscle.msm-TS - EjiI . mi. *. 2 output in the perfused hind-limb. The rate of ,4C02 output was s t i l l rising at the end of the 2Rates o f leu cin e oxidation in the perfused h in d -lia b : e f fe c t o f p ro te in -fre e fe e lin g and va ria tio n in the aaino acid concentration o f the p erfu sate. Means * SD.S I B0 0*Dp«S)AUDIO ACIDm m m m mKJSCLS SR or WEIGHT JCSCLE ICT ( f ) (d ja/m ole)1^C02 OOTiVT (dps x 1o V *in )RATE OF ISJCDIE OHDATIOH (/m >U»/bx) T o ta l per g nuscle per 81^180g ra10 high 54.2 4 2.4 90 4 24 7 .0 1 0.6 4.®5 1 1.8 0.10 i 0.02 7.94 4 2 .110 low 50.1 144 11 .6 4.82 0.10 7.800 high 49.9 4 4.0 105 4 96 6 . 4 4 0.7 9.99 4 1.6 0.06 1 0.09 6.59 4 1.70 low 55 .9 1 9.1 122 1 29 4.0 4 0.4 2.09 4 0.6 0.04 4 0.01 2.96 4 0.7* d if fe r s s ig n if ic a n tly from Group 1 t p > 0.05.* perfusate contained 80.4 x 10 ^ Apn/ain - leu cin e.95(group 3) led to a f a l l in absolute oxidation which was not s ta t is t ic a l ly .significant. (Table 17). I f , however, the lisibs were perfused with Low aaino acids, the output of '^CO^ decreased despite an increase in specific radioactivity of in trace llu la r free leucine (Table 17, ) . The absoluterate o f oxidation was reduced quite sign ificantly from 3.99 /•■oles/hr to 2.03 jtaaoles/hr on altering the levels o f anino acid and insulin. This reduction f i t s in with the previous work carried sut in vivo and in v itro .The contribution o f nuscle to total body leucine oxidation SMy be estimated from the data in Table 17 i f several assumptions are made. This is considered in Section HID. Skeletal nuscle, which forms approximately 8 o f the hind- limb preparation, was assumed to be the sole contributor to leucine oxidation by the hind-limb. Possible contributions by bone, connective and nerve tissue were not considered although recentJy Hu.,, Jurm in ic A Reid (1975) have published data on the oxidation o f branched-chain amino acids by nerve, muscle and aorta. Since adipose tissue is also known to oxid ise leucine (Rosenthal et a l, 1974) the major fa t pads overlying the psoas muscles were removed during the surgical preparation. The vessels supplying the skin were a lso ligated to minimise any oxidation by this tissue. The success of these measures is borne out by the results on protein synthesis in the hind-limb, in the following section.C (V i) PK0TE1N SYNTHESIS RATES IN Tiff: PERFUSED HIND-LIMBProtein synthesis rates in skeletsl muscles o f the perfused hind-limb were calculated by the equation given in Section II IB (iv )96¿•rived by O arlick ct al (1973). The pool sizes o f free leucine in pe rfu sa te and gastrocnemius muscle are show in Table 18, and the synthesis rates in Table 19. The fractional synthesis rate (PSR) of protein in skeletal muscle of the normal (0.10 NDpsE) hind-limb perfused with High amino ac ids (Table 19, Group l ) was remarkably s im ilar to that of sk e le ta l muscle protein in vivo (Table 11). Furthermore, when limbs from rats fed the protein-free diet were perfused with Low amino acid levels (group 4 ) the PSR of protein in gastrocnesius muscle was reduced from 8.85^ D * in the normal limb to 4.69£ D*'. Again, this value was sim ilar to the rate observed in vivo in animals fed a protein free d iet. This reduction in fractional aynthesis rate brought about by protein-deprivation is in agreement with the findings of G a rlick ot al (1975) carried out in rata in vivo but infused with [(J1 )-tyrosine. This agreement with resultr in vivo rein forces the earlier claims that the preparations were viable fo r a t least 2 hours. It is o f interest to note that when eithe r nutritional group was infused with Low amino acid levels the pro te in synthesis rates appeared to decrease (Table 19, groups 2 and 3), but lesa sign ificantly in the protein-free group (4 ) . This is a l l the more interesting since recent work ers have suggested the possible role o f leucine in the regulation of protein synthesis in skeletnl muscle (Fulka et a l , 1975| Buse A Reid, 1975). However, no claims are made here as there were only 2 observations in the 10£ NDptE, Low amino acid group. The hypothesis w ill be discussed, however, at a later stage.&T1BLS 18 Concentrations o f f m leucine in perfusate and la intracellu lar flu id (ICP) o f gaatrocneaiua ouaelo after 2 hours' perfusion o f the hind lla h , and in plasna and anscle jr. tItq. Means ♦ 3DCroup l o . la Diet ialr.o sold Leucine pool e lse (•(n o le a / a l or uaolee/g. tissue wet weight)perfused group concentrationM m p ii) Perfusate or plassa Muscle ICF Ratio ICP/plaoaa1 5 10 high 158.2 ♦ 2 5 . 6 261.5 ♦ 24.7 1.89 1 0.52 3 10 low 98.0 ♦ 8.6 242.5 ♦ 35.1 2.47 ♦ 0.43 5 0 high 125.4 i 30.1 223.7 ♦ 29.2 1.78 ♦ 0.34 4 0 low 88.1 ♦ 19.0 1 9 5 .7 * 8.0 2.22 ♦ 0 .5in elrp7 10 - 104.5 i 10.4 170.6 ♦ 15 .4 1.63 1 0.39 0 - 76.7 i 12.4 160.2 ± 1 1 .7 2.09 ♦ 0.4101leucine oxidation ratea in total skeletal anísele are estimated to fa ll from 166 pinoles leucine/day/lSOg body vt (group 1) to 69 liusoles/day/l80g body vt (gro»-> a (Table 21).In v ivo , the total body leucine oxidation f e l l ir^si 766 junóles/ Isy/lSOf body vt to 486 ^ im oles/day/tbody vt in identical groups of animals (Table 13). Therefore, the reduction o f 97 /imoles/day in the perfused hind-limb preparation (skeletal muscle) represents at least of the fa l l in leucineoxidation seen in vivo on changing from a 10£ NDp:E diet to a Of MDp :E d iet. Since this is an under estimate (Section I IIC (v ) of skeletal muscle leucine oxidation, i t is evident that muscle is one o f the major contributors to the control of leucine oxidation in tiaras o f dietary stress. These figures also suggest that in the ve il fed group skeletal muscle is contributing at least 22% o f total body leucine oxidation.Vhen the hind-limb from v e il- fed rats is perfused vith lov amino acids (group 2 ) the oxidation rate increases s ligh tly (Table 21) . i t limbs tra e rats fed Of NDp:E diet are perfused vith high levels of amino acids (groigi 3) then leucine oxidation rates are greater than vhen the amino acid concentration of the perfusate is lov (group 4 ).fa » * » » "The estimates shovn in Table 21, of the uptake o f leucine into total muscle protein, are derived from flux measurements on the hind-limb on the assumption that a l l ( ,4C ] leucine vhich is not oxidised is taken up into protein. The results are given in terms o f panoles leucine/lSOp body veight/day. These can be converted into fractional rates o f muscle protein synthesis.on the assumption that muscle is 45^ o f body weight, and contains 20^ protein. Thus we have 3 ways o f calculating the rate of protein synthesis in muscle.A. From measurements o f la be llin g of protein and free leucine in gastrocnemius a fter constant infution in the whole anisMl (Section l l I B ( iv ) .B. From the same measurements, a fter perfusion of the hind-limb (Section II IC (v i ) .C. From measurements of flux and oxidation in the perfused hind-limb (th is section ).Methods B and C, although they are based on the same preparation and although both re ly on determination o f the SR of free leucine in gastrocnemius, are independanti in B the additional information is obtained from the SR o f protein, whereas in C it is obtained from aieasuring the oxidation.The resu lts o f this comparison are shown in Table 22. The Agreement is reasonable, suggesting that the assumptions on which the various calculations are based are not too much in103«TABU 22 Cospirison o f protoln »ynthaili rato* in ikalatal «usci» aitinatad (A) in tìvq( l ) in tka faatrocnanina s u o lo o f tha parfuaad hlnd-linfe and (C) fr<* fio * rmtaa ln Um bind-linfe.Dlatary«roupPiotala ijm thaiii rata (M *1)(W lpl ! ) ABi£ TlTQ gaatrocnanlui n u d a (bind linfe)tota l nuscla (U n i linfe)10 5.52 5.00 8.650 5.97 6.80 4.65106■PA transfprase activityIn agreement with moat other workers, the highest aminotransferase ac tiv ity was found in skeletal muscle. Bothfasting and low protein feeding led to an increase in the skeleta l muscle aminotransferase a c it iv ity , whilst an increase in the liver ensyme ac tiv ity was observed only in animal a on a low protein d iet.Ichihara, Noda ft Ogawa (1973) have suggested that the step involving transamination may be the rate-lim iting reaction in the oxidation o f leucine, particu larly in the l iv e r . This supported Krebs' suggestion (1972) that the aminotransferase could be rate-lim iting through Ksi control. The value often quoted for the Km o f leucine is 3 .SaM, obtained from seasure- mente on hog-heart (Ichihara ft Koyama, 1966). The Km fo r BCA aminotransferase in rat skeletal muscle has never been published, but the present work gives a value close to 0.4 mM t o r leucine (Table 2 ). This is very much closer to the Km value of the dehydrogenase than was previouuly thought. I f the ensyme were to decrease in protein-depeletion then this would iwpresent an additional 'course' control over amino oxidation. Oi the other hand measurements in vitro show that the aminotransferase ac tiv ity is many times that of the dehydrogenase. I f these results hold in vivo the ac tiv ity of the dehydrogenase must be rate-1imiting.The literature does not agree on the effect o f either starvation or protein depletion on leucine aminotransferase ac tiv ity in rat tissues. McParlane ft Von Holt (1969 b ), Krebs (1972) and Adibi et a l (1975) have a l l shown reductions in the ensyme in tissues from protein depleted rats. Moreove r, Me Pa r 1110?Von Holt found that adaptation o f this enzyme occurred in the HVtS:»cX>-C'*.A.r>a(fraction. This would agree with Shirai et al (1971) that Enz I I (mainly mitochondrial) was more responsive to induction than Enz I (supernatant). Both Adibi et al (1975) and Krebs (1972) found that protein depletion did not a ffe c t the aminotransferases in kidney.On the other hand, Mimura et a l (1968) found that a pro te in -free diet led to increases in aminotransferase ac t iv ity in muscle, kidney and l iv e r but not in intestine. Reeds (1974) observed only slight increases in the enzyme in skeleta l muscle and a marginal decrease in the liv er enzyme ac tiv ity . In the present work, Mimura's observations have been confirmed in rats fed a protein-free d ie t . However, i f rats were fed a low protein d ie t . Just sufficient to maintain body weight, then only muscle aminotransferase increased. Recently, Peatheraton A Horn (1973) observed no effect on leucine aminotransferase ac tiv ity in chick skeletal muscle, l iv e r or kidney following 48 hr starvation or a protein-free meal. On the other hand, both in A d ib i's work (1975) and in that reported here starvation led to an increase in muscle aminotransferase.Evidently, therefore, the literature is in a state o f confusion. Mich of this may a r ise from the fact that we are dealing with several isozymes ( I - I I I ) and «hat compartmentation o f these isozymes may lead to different responses, depending on the 't r ig g e r ' agents and 'penetration o f compartments'. Id ea lly , one should study the effects o f protein swlnutrition on individual isozymes within compartments such as mitochondria and c e ll cytoplasm. McParlane's work (1969 b ) goes some way to achieving this, but no106comprehensive study has been Bade- up to the present time.BCA Dehydrogom.seIn the present work, skeletal au sc le was estimated to have the highest total tissue dehydrogenase ac tiv ity . Both fasting and protein-free feeding led to a decreased ac tiv ity o f the dehydrogenase eniyne in skeletal m uscle. In l i v e r ( however, fasting increased the encyme a c t iv it y whilst a low protein diet, suffic ient to maintain body w eight, did not a ffe c t the entyse ac tiv ity . Branched-chain * —oxo acid dehydrogenaseactiv ity has a wide tissue d is tr ib u t io n in the rat and other animals. So fa r , i t has been demonstrated in liv e r , kidney, heart, skeletal muscle, skin, lung, in testine , brain and white blood ce lls (Connelly et a l 196R| Volhueter k Harper, 1970| Reeds, 1974| Danner et a l , 1975). The present work demonstrates the presence of OC -oxo isocaproic ac id dehydrogenase and x -o x o isovalerate dehydrogenase in rat sk e le t a l muscle. P rio r to this moat workers were unable to demonstrate any dehydrogenase activ ity in rat skeletal muscle (C on ne lly e t a l , 1968» Volhueter i Harper, 1970). The fa ilu re o f Volhueter i Harper to find this ensyme in muscle may have been the r e s u lt of their use o f the Polytron homogeniser for the preparation o f muscle extracts, as my investigations showed that this technique causes a tota l loss o f ensyme activ ity . Presuswbly, soni cation disrupts the ensyme complex from the outer face o f the inner mitochondrial wall where i t is known to be located (Johnson k Connelly, 1972). By using gentler methods of homogenisation i t has been possible to show that OC-ktC dehydrogenase w ith in the total mass o f erasele may be as much as thirty times that found in whole liv e r . Reeds(1974) also demonstrated Ot-KIV dehydrogenase activity in rat109skeleta l muscle at much the same time. He, too, homogenized the muscle in Dun 11 glass tissue grinders.Muscle has the greatest total enzyme activ ity and is considered to be the major s ite o f BCA oxidation. Recently, Beatty et al (1974) reported that there was no difference in the output on 14C02 from labelled leucine in incubated red or white skeletal muscles. However, an exploratory survey of d iffe ren t muscle types demonstrated the p o ss ib ility of varied d istribu tion of enzyme ac tiv ity (Table 3). Thus the greatest dehydrogenase ac tiv ity was found in the p lantaris and lower a c t iv it ie s in the Extensor digitorum longus (EDL) and soleus muscles. There exists in muscle another system for the doamination o f amino acids, other than the aminotransferase-dehydrogenase system already discussed. This is the scheme proposed by Lowenstein (1972) in which a 'purine nucleotide cycle' catalysed by the sequential ac tiv ity o f the enzymes adenylosuccinate synthetas«. (I .C .6 .3 .4 .4 ) , adenylo succinate lyase (B .C .4 .9 .3 .3 ) and AMP deaminase (E .C .3 .5 .4 .6 ) brings about the deamination o f aspartic acid. Operation o f the cycle (Tornheim ft Lowenstein, 1972) appears to be k inetica lly linked to glycolytic ac t iv ity (Tornheim ft Lowenstein, 1973). Lowenstein (1972) demonstrated an inverse relationship between the ac tiv it ies o f glutamate dehydrogenase and AMP-deaminase in a number o f rat tissues.Turner ft Pern (1974) have shown that AMP-deaminase ac t iv ity is lowest in the soleus, intermediate in the EDL and highest in the plantaris muscle. The present observations o f the BCA dehydrogenases seem to fo llow a sim ilar pattern. Turner et a l (1974) also suggested that the reciprocity between the ac t iv it ie s of AMP deaminase and glutamate dehydrogenase extended to different11 0types o f skeletal muscle. They showed that protein restriction resulted in decreased AMP-deaminasc ac tiv ity in plantaris and aoleus and had no e ffe c t in the EI)1.. Relatively smaller decreases in AMP-deaminase activity were found in soleus and plantaris when a protein -free diet was fed. This response agrees with the observations made earlier, that a protein-free diet leads to a reduction in BCA oxidation, but a low protein diet produces an ever greater reduction. Thus i t seems that the conservation of branched-chain amino acid oxidation in rats fed either a low protein or protein-free diet is «ssociated with the lowering o f both AMP-deaminase and BCA dehydrogenase activ ities ia skeletal muscle.Fate o f theft, -oxo acidsIn considering the ac tiv ity o f the ensymes in vivo, it has to be borne in mind that measurements were made on crude homogenate preparations and may not bear any physiological significance.It is noteworthy that urinary excretion o f # t-o io isocaproate is increased in the protein depleted rat (McParlane A Von Holt,1969 a ) . This would suggest a decreased breakdown o f this branched-chain h. -oxo acid and a consequent increase in its pool • i t t . Reeds (1974) suggested that thsa-eiia acid may sw>ve from muscle to liv e r , where further oxidation or reamination could occur (Valser et a l , 1974). Excess would presumably be excreted in the urine. The.-e are few reports on the concentration of branched-chain et-oxo acids in plassw (Tanaka et a l , 1972) and certainly none which give concentrations in the in tra-ce llu la r pool of rat tissues. The in vitro entyme measurements show a transient r ise in the BCA aminotransferase ac tiv it ies in both icle o f protein-depleted rats. At the same time.iver and111the dehydrogenase ac tiv ities were decreasing. Presumably, the production ofOc-oxo acids might exceed the capacity o f the dehydrogenase. Moreover, this enzyme is known to be inhibited b y c * o - a * ^ * (Volhuetcr e t a l , 1970). This in i t s e l f might act as a feed for ward in h ib it ion of the Irreversib le oxidation o f the BCAs in muscle. In starvation, where increased oxidation is observed, i t i s only ¡1% liver that the dehydrogenase activ ity is increased, together with the ami no.ran»ferase activ ity . This would seem to support Reed'shypothesis that the oc-oxo acid might pass from muscle to l iv e r . However, this needs to be clarified by measurements o f plasma and tissue intra-ce llu la r concentrations o f ck. -oxo acid in control, starved and protein depleted ra ts , since Tanaka et a l , (1972) found l i t t le or no increase in the ot^-oxo acids of starved rats.(11) THE CHOICE OP LABELThis thesia is primarily concerned with the oxidation of the branched-chain amino acids and in particu lar o f leucinv. There have been d iffering opinions aboui the degree o f adaptation in BCA oxidation in malnourished rats which have arisen as a resu lt of the use of d ifferent labelled forms of leuc ine and va'ine.Me P a r lane A Von Holt (1969 a ) found that 14COz output from DL- leucine, labelled in the 2C position, was decreased in rats fed a 2% casein diet for eight weeks. Neale (1971* 1972), however, could not detect any reduction in the oxidation of U,4C labelled leucine, valine or lysine or of mixed amino a c id s when given to rats fed a 1 % casein diet for 15-17 days. N eale (1971) administered the amino acids either by the in tragaatr ic or intravenous112route. He found that protein depletion led to an actual increase in 14C0., output from a l l amino acids except valine.The route of entry made no sign ificant d ifference to the output of in either the control or protein depletedgroiqis. This was in agreement with work by PicoVkft Taylor— Roberts (1969), who compared the turnover rates o f 15N-glycine by intragastric or intravenous routes in two children, and found no sign ificant difference. Neale concluded that the BCAs in particular could not be conserved in protein depleted rats .His later studies (1972) supported this hypothesis since to ta lly •viscerated rats previously maintained on a protein -free d ie t , were unable to reduce their oxidation o f injected valine or mixed amino acids compared with eviscerated rats previously given a high protein d iet . This suggested that the extra-hepatic tissues are unable to adapt in protein-depleted rats.McParlane's choice o f M.[2,4c ]-leucine (1969 a ) may be c r it ic ised on two accounts.Firstly , D-amino acid oxidase is known to be present in most tissues (Meister, 1965 a ) and any adaptation in response to reduced protein intake may re flec t adaptation in the D-amino acid oxidase as well as in the catabolic ensytnes involved in the normal pathway o f leucine degradation. Indeed, further studies by McParlane g co-workers (1969 b) did show a reduction in activ ity of the D-amino acid oxidase in liv e r . Secondly, with the label in the 2C- position the acetyl CoA moiety in which the label appears could theoretically be syphoned o f f into fa tty acid synthesis, cholesterol synthesis and the TCA cyrlg . I f s ign ifican t amounts of ,4C are retained in these other compounds, measurement o f the output o f 14C0j w ill underestiswte the true extent o fleu<113There »re aim ilar objections to the uniformly labelled L-leucine or L-valine that Neale used to investigate adaptation o f the BCAs in malnourished rats (1971, 1972). A fter decarboxylation, the remaining Catoms o f the carbon skeleton could be shunted via hydroxy-methyl-glutaryl CoA (HMGCoA) into cholesterol productioni via acetmacetate into fa t metabolism; or via acetyl CoA into the TCA cycle. Again i f any of these pathways are active, they might obscure a reduction in the in it ia l irrevers ib le oxidation o f leucine at the step when ¡A.KIC is decarboxylated.Reeds (1974) s p ec if ica lly examined the effect o f the position of the label by comparing the oxidation of valine withthat of L Jl' 4cJ -va1ine in rats fed a low protein d iet identical to that employed by Neale (1971). Reeds showed that with .uniformly labelled valine there was no difference between protein- depleted and control animals in the output of ^CO^, either in vivo or in v it ro . The resu lts, therefore, were in agreement with those o f Neale <»9Tf). However, when identical group« o f rats were given L £ l '4cJ-valine the excretion o f was reducedin the low-protein group. This supported my observations at much the same time, but with L fl 14cJ-leucine. Reeds argued from the theoretical viewpoint th »t " if some o f the non-carboxylic label in U,4C valine is retained in protein, glucose, fa t or cholesterol, then it follows that an estimate o f valine catabolism with l [ i 14c J- valine w ill always be higher and should never be lower than an estimate with U,4C -valin e". He showed this to be true in v it ro , but in vivo in weanling rats and in protein-depleted rats the estimate o f va lin e catabolism with fl 14C -^va 1 ine was lower than with uniformly labelled valine. Two explanations were o ffered.1) Tho decarboxylation of ocKIV was not rate-lim iting.2) That M C02 formed by the decarboxylation o f ocKIV enters the bicarbonate pool at a slower rate than labelled CO^ formedin the TCA cycle, or is preferentially r e u t ilis e d in C02 fix a tio n .The bicarbonate pool has been estimated in the rat to turn over with a h a lf - l i f e of 12$ minutes (M illward; 1970). Measurements o f the turnover o f CO^ with l ' 4C-leucine (Section IIIA) gives almost identical results, suggesting that the carboxyl C does not enter the bicarbonate pool at a slower ra te than the other C atoms o f the ami acid.Therefore Reeds' observation remains unexplained.This thesis was also concerned with measuring quantitatively the rate o f leucine catabolism in both the perfu sed hind-limb and the whole animal. The constant infusion method (Vaterlow ft Stephen, 1968| Garlick ft Marshall, 1972) a llo w s us to do th is .The overall equation of the balance of amino acid flow into and out of the plasma compartment is l Q - S ♦ C “ B ♦ I(Flux * Synthesis + Oxidation ** Breakdown * Input)The rate o f amino acid oxidation (C) can be determined from the rate o f excretion o f ,4C0o at plateau. The proportion of the infused dose excreted as 14C02 gives us the proportion of the flux oxidised. The equation assumes that the only pathways of amino acid u tilisation are uptake into p ro te in and oxidation.Moreover, as already pointed out, the measurement of '*C02 excretion w ill only give a correct estimate o f oxidation i f «( l ) no products o f oxidation are retained in compounds such aa fa t , and (2 ) no ,4C<>2 is retained in or taken up from theii*1 1 5bicarbonate pool.As we have seen, by using an amino acid labelled only in the 1-C position the f i r s t requirement is met since on oxidation the 1-C atom passes d irectly into iho bicarbonate pool. Measurement o f the rate of excretion of ,4C02 from the bicarbonate pool (Section IIIA ) shows that the second problem is also overcome in the rat when we use this form of labelled leucine.Therefore, in conclusion, theoretical considerations wero much in favour of L Jl *4cjlabe lled BCA being used both in vivo and jin vitro throughout the experiments described in this thesis.In it ia l experiments with DL [l Mc]-leucine were carried out, but Reed's (1974) confirmation o f the problems involved in the use o f UM C label made any further investigation with this particular label unnecesrary.( i i i ) The DL o rU -i»o so r in Measurements of OxidationMe Par lane A Von Holt (1969 a ) observed that 40* o f a dose o f M.[214cJ-leucine was excreted as ,4C<>2 in control rats and that this figure was reduced to 10* in protein-depleted rats. Similar results were obtained in the present work when rats fed either a high or a low protein d iet were injected in tragastrica lly with a pulse dose o f DL^I^4cjleucine. Both groups had higher rates of 14C02 excretion than was obtained with the L-isomer (Table 5).The higher rates o f excretion observed with DL- mixture of isomers My resu lt from the high activ ity o f the D- amino acid oxidase ensyme in liv e r mitochondria (McFarlane A Von Holt, 1969 b ).MeFarlane et al found that ,4002 production from D |l,4c]-leucine was twice that o f L [l ,4c ]-leuc ine and that the spec ific activity116----- o f D- amino acid ox idare was aj>j>rc ximnt e ly 10 fo ld that ofleucine aminotransferase. Further evidence indicating the errors which may be caused by the D-isomer waj obtained on investigating the effects o f fasting in rats fed high or low protein diets. Vith DI,-leucine we could not detect the increase in oxidation o f leucine nonsally observed with the L-isoner (Section I IA ( i v ) ) .I f accurate measurements o f production are to be madefo r the purpose of estimating flux rates by the constant infusion method (Vaterlow A Stephen, 196S) then i t is essential that the L-isomer is used. For these reasons, l [ i leucine was used in a l l subsequent studies.( iv ) Tissues Oxidising LeucineThe a b ility to transaminate and decarboxylate leucine and the other BCAs is widely d istributed . Tissues which can do this include the kidney, liv e r , heart, muscle and brain (Dawson A H ird, 1967| Clarke, 1957| Buse A Ruse, 1967| Johnson, Herring A Field , 1961) as well as human white ce lls and skin fibrob lasts in culture (Dancis, Hutsler A Levits, 1961). Adipose tissue is nlso capable of oxidising leucine (F e lle r A Feist, 1962) and Rosenthal et al (1974) suggested that in humans, adipose tissue was one of the major extra-hepatic sites fo r leucine metabolism, concerned mainly with the biosynthesis o f sterols. Recently, oxidation o f leucine has a lso been observed in sc iatic nerves Kertog et a l , 1974) and in the isolated aorta o f the rat (Buse, Jtirainic A Reid, 1975).Despite the overwhelming desKinatration o f the widespread ab ility117also dogmatically stated that skeletal siuscle i s the major s ite o f leucine oxidation. As discussed e a r l i e r , this stems from the work o f M ille r (1962), who demonstrated that extra hepatic tissues were as capable o f oxid ising leuc ine as liver. However, his preparation would also have included skin, adipose tissue, peripheral nerve, brain, lung and kidney as se ll as skeleta l muscle. Manchester (1969), then Meikle * Klain (1972) Mid Odessey ft Goldberg (1972) argued that i f the metabolism of diaphragm (the tissue studied) was representative o f that of skeleta l muscle in vivo, then muscle was p robably the major s ite fo r C02 production from leucine, since ske leta l srascle comprises approximately 49^ o f body weight o f the rat (Nunro, 1969). A more accurate approach to the estimation o f o x id ation rates in v ivo , and the isolated perfused hind-limb was attempted in the present work and w ill be discussed at a la t e r stage. However, in the context o f the p resent discussion i t is worth noting the work carried out in vitro on the regulation o f BCA oxidation in skeletal muscle, diaphragms, heart and liv er .( r ) a m u r n i E f f e c t , on USA o t ii to t lo n - I n s n llnManchester (1969) found that when rat diaphragms were incubated in the presence o f insulin (100 mCJ/ml) there was a consistent but small stimulation o f oxidation o f L^I * *c jle u c in e . However, neither Meikle ft Slain (1972), using 30 mU/ml in su lin nor Buse, Biggers ft b:se (1972) with 1.0 mU/ml insulin were ab le to repeat Manchester's observations on incubated diaphragms. Insulin did stimulate production from leucine by hearts obtained fromrats a fte r a 48 hr fast and perfused without g lucose (Buse et a l , 1972). This may have represented stimulation o f amino acidtransport in to 11s by in s u lin (M anchester, 1970),118Mi 11 ward et al (1974) »hovel that in ra t » fasted fo r 72 hour» tha in tracellu lar concentration o f BCAa increa»ed 4-5 times.At the same time the plasma insulin concentration decreased by 46*. Oxidation o f BCAs was not measured in theae experiments, but i t i i reasonable to suppose that i t was increased in the starred rats . In this case, therefore, an increase in oxidation would be associated not with stimulation by insu lin , but with increased a v a ila b ility o f free amino acids in the in trace llu la r pool, derived from protein breakdown.feinephrine and GlucagonBus», B igger», Drier 6 Buse (1973) found that stimulation o f BCA oxidation by epinephrine in diaphragm and by glucagjn in heart was only demonstrable in tissues from fasted rats , and only during incubation or perfusion without glucose or pyruvate. Moreover, the fact that perfusion with 5.5 mM glucose suppressed the stisnilation o f branched-chain amino acid oxidation by epinephrine or glucagon suggested that hormonal stimulation o f BCA oxidation may not occur under physiological conditions. However, hepatic gluconeogenesis from alanine is stimulated by glucagon (M allette et a l , 1969). A cycle involving the branched- chain amino acids may complement the alanine cycle. Under conditions when the hepatic uptake o f alanine is stimulated, the hepatic output o f BCAs is increased. In muscles, stimulated oxidation o f branched-chain amino acids cosplesents the release o f alanine, the carbon skeleton o f BCAs serving »■ an energy source for muscle c e lls and the amino group fo r the transamination of pyruvate to alanine. It is not suggested that this would bequantitatively important.119CorticoaU Toid .iGlucocorticoids are known to cause protein catabolism in peripheral tissues and thus to cause an increase in the pool sice of free amino acids in the liv e r . Although work has been carried out on the induction by corticosterone of liver and kidney leucine transaminase activity (Shirai & Ichihara, 1971) it is only recently that oxidation o f leucine by incubated skeletal muscle has been studied in vitro (Ryan et a l , 1975). These workers deawmstrated that 24 hr a fter corticosterone injection into a rat , teased strands of the transversus abdominusmuscle on incubation oxidised greater amounts of l.£l -leucine i I • •than controls who had not received corticosteroids. However, in the same group o f experiments, Ryan et al (1975) demonstrated that teased strands o f muscles from 24 hr fasted rats oxidisedless leucine than unfasted controls, which is contrary to the • • , - saccepted view o f the effects of fasting on leucine oxidation inU. Imuscle. This work obviously needs repeating before one mighter »n..conclude that corticosterone has a regulatory role in leucine oxidation during fasting.(v i ) E ffect of Fasting on RCA OxidationThe experiments with animals fasted for 4® hr show that oxidation oT'leucine is increased in both the groups fed a normal and a low- 'protoin diet. The sequence of events appears to be as fo llows. Increased amounts o f free leucine are liberated in muscle, as a resu lt of reduced protein synthesis and increased protein breakdown (Hi 1lvard, 1970). Free leucine levels in muscle do r ise , but this rise is not progressive, nor very great. This means that the increased production o f leucine must be balanced by anincreased rate o f removal.120Measurements o f A-V differences in the forearm during fasting show l it t le increased output o f leucine as such. Most o f the extra output o f amino acids is in the form of alanine and glutamine (F e lig et a l, 1969). Presumably amino groups from other amino acids, including leucine, are transferred to alanine and glutamine by transamination and by the action o f glutamine synthetase. Perfusion experiments (Ruderman A Lund, 1972) showed that addition o f leucine to the perfusate led to an increase in alanine and glutamine and that glutamine alone was increased when NH4C1 (5mM) was added. Alanine synthesis is catalysed by glutamate-pyruvate aminotransferase and glutamine synthesis by glutamine synthetase. I f L—methionine D, L-aulphoximine (a specific inhibitor of glutamine synthetase) was present, the increase in glutamine release induced by leucine was diminished ky 50JC. Glutamine rather than alanine appeared to be the major vehicle of nitrogen transport from muscle to other tissues .In these circumstances, one might expect an increase in ac tiv ity o f the enxymes catalysing these reactions - at least in muscle (a ) the aminotransferase and (b ) the dehydrogenase. Transamination , with the donation o f the amino group from leucine to alanine or glutamine (Pelig k Wahnm. 1971) would be fa c ilita ted by the high concentration of aminotransferase present in muscle (Toung, 1970). Tills transamination seems to occur more read ily than is normal, because, despite the i*t breakdown of muscle protein with the production of sere leucine, there is no great accumulation of free leucine within the muscle. The ensyse experiments with fasted aniamls showed that leucine aminotransferase was increasing in activ ity in both muscle and liver. In contrast, the activities o f liv e r and kidney leucine aminotransferase( Vo] hueter ft Harper, 1970), the f i r s t ensyme of the leucine12 1are unchanged a fter fasting .More recently, Adibi et a l (1979) measured leucine aminotransferase ac t iv it ie s in liv e r , skeletal muscle and kidney from starved rats. A fter 12 hr o f starvation both muscle and kidney ensyme ac tiv ities were s ligh tly reduced. When starvation was prolonged for a fu l l day, leucine aminotransferase increased approximate! y two-fold in both tissues.A 9-day fast resulted in an additional increase in spec ific ac t iv ity of the ensyme in muscle. Throughout the prolonged starvation period, leucine aminotransferase ac tiv ity remained unaltered in liv er . These changes may be functionally important..The ensyme experiments vith fasted animals showed that both liv e r and musclest-oao acid dehydrogenase ac tiv it ie s altered s ign ific an tly within 48 hr (Table 6 ). Muscle ensyme ac tiv ity was diminished, whereas liver ensyme ac tiv ity increased. Formerly, Voihueter ft Harper (1970) showed that the activ ity o f l iv e r •<. - oxo isocaproate dehydrogenase, the second ensyme in the pathway o f leucine catabolism, and the ac tiv ity of skeletal muscle •«*<* CoAtransferase (Williamson et a l , 1971), the f i r s t ensyme involved in acetoacetate u tilis a t io n , increased during fasting . Ilia former agrees with the present observation that *<. -oxo isocaproate dehydrogenase activity increases during fasting in the l iv e r (Table 6 ), I f the step o f decarboxylation were not rate-lim iting, then an increased provision of substrate would Increase oc oxidation despite the lowered dehydrogenase ac tiv ity seen in muscle. Any lim itations in decarboxylation could also allow the excess(xo acid to pass f r i ile to l iv e r , where the increased122dehydrogenase a c t iv ity would aid it s further oxidation. This would ce rta in ly be a mechanism whereby liv er could derive energy from the carbon skeleton o f br»niched-chain ami..o acids present in high concentrations in muscle protein. However, since fa st in g does not normally lead to a large increase in the plasma concentration o f the BCA « . -oxo acids (Tanaka et a l , 1972) and the capacity o f liv e r oxid ising oxo acids is lim ited, i t is possible that muscle plays an important part in the increased oxidat on o f leucine during fasting .Clarke (1957) and Manchester (1965) have shown that heart and isolated rat diaphragms were capable o f oxidising leucine to give rise to CO^. Goldberg's (1972) experiments with diaphragm also suggested that the ec. -oxo acid o f leucine is oxidised in muscle and the incubation experiments (Table 6 ) provide further evidence o f the oxidation of leucine to ei rbon dioxide by sniscle. Subsequent work in Goldberg's laboratories (Odessey A Goldberg,1972| Goldberg ft Od«Ssy, 1972) showed that appreciable oxidation o f leucine occurred in the dark soleus and pale extensor digitorum longus muscles. Moreover, diaphragm catabolised leucine at rates sim ilar to l iv e r s lic e s but several fo ld less active ly than epididymal fa t pad or kidney and brain s lic e s . Oxidation of L^I 14C^-1 aboil ed leucine, valine and iaoleueine increased three to five fo ld in the diaphragms o f animals fasted for 3 days.Protein synthoris during this period was 50% lower than control values. Tbs f a l l in amino acid incorporation was evident within one day of fasting, but amino acid oxidation did not increase un til the second day. Ve have previously seen that leucine aminotransferase was increased in muscle (Volhueter ft Harper, 1970 | Adibi et al 1975) by day t . o f a fa st and that by the second day the ff(. -oxo-— Aisocaproate dehydrogenase had increased in liv e r but decreased in muscle (Table 6 ). The km of the dehydrogenase a c t iv it y in l iv e r mitochondria has been shown to be 0.2 sunol/l (Volhueter A Harper, 1970). Since the muscle enzyme has ap p ro x im a te th e same km (Table 2 ), the the concentrations of leucine in muscle are the approximate range in which the oxidation rates w i l l be deteimined by the leucine concentration. It is un likely that the intra-ce llu la r pool o f amino acid is homogenous- so that variations in leucine concentrations greater than those seen for the whole tissue may occur at the precursor site fo r oxidation . Thus an enhanced absolute rate o f oxidation may occur in muscle in starvation despite a fa l l in the apparent ac tiv ity o f the enzyme assayed in v it ro . ,( v i i ) E ffect of Feeding l.ow Protein Diets on BCA C-jdati onStephen (1968) and Vaterlow (1968) have stressed the importance o f econoqy and recycling o f nitrogen in the mechanism o f adaptation to low protein intakes. Neale (1971) later suggested that the limiting factor may be the animal's ab ility to reduce the oxidation of the carbon skeleton of essential amino ac ids. As mentioned ea rlie r, he found that the branched-chain amino acids in particular showed no reduction in oxidation in protein-^lepleted ra ts .However, a number o f studies provide evidence o f adaptive change in amino-acid oxidation. Tamsshita A Ashida (1969) | » » « U,4 C- ly sine intraperitoneally to rats on a lysine-free d iet and found a sign ificant reduction in output. McFarlane A Von Holt(1969 a ) found a narked reduction in the oxidation o f Dl. [ ' <- Jucine and U,4C -phenylalanine given iatraperitoneally to ratale i12<*on i 2?S casein d iet. There was no change in the oxidation ofpresent work has confirmed Me Fur lane k Von H olt 's observations. Vhen rats that had been fed a 3.5'/ NDp:K diet f o r 3 weeks wereto controls on a normal d iet. At much the same time, Reeds (1974) a lso confirmed McParlane's work, using l [ i ,4C^ valine.leucine, valine, lysine or phenylalanine were constantly infused intravenously fo r periods of up to 4 hr. there was no difference in the proportion o f dose oxidised to CO^ in rats adapted to aVate r i ow, 1974 a ) . More recently, Neale (1975) examined thegiven high casein or low casein d iets (250g/kg and 5Ug/kg). At this protein intake, the body-weight of the la tte r group was maintained constant for periods o f 7 - 9 days. Without prior fasting overnight (as in his previous experiments) the rats were infused with the labelled valine for periods o f up to 7 hours, with continuous collection of ,4C02 at half-hourly intervals.The provision o f maintenance levels o f protein now produced an overall reduction in oxidative catabolism of U,4C -valine to ,4C0^. Neale, however, s t i l l sutintained that a protein-free diet caused a breakdown in the adaptive process, resu lting in a high rate of loss o f essential amino acids from the body (Neale kthe -essential ids all glutamine. TheHowever, when U- used there reduct ion*4C<>2 output in protein-depleted rats. This second observation confirmed Neale 's work (1972) who later found that even i f U*4Cprotein—free d iet compared with those »trol d iet (Neal.catabolism of U,4C valine in adult male Vistar rats (250-300g)Vaterl 1974 b )However, in the present vo.-k a net reduction in the oxidation o f I , [l14cJ-leucine has been observed in rats o f a l l ages fed a protein-free d iet. This has been a consistent observation in rats given either a pulse intragastric dose or a constant ta il rein infusion| and in the perfused hind-limbs o f pr „ein-depleted rats. These observations have been supported by para lle l measurements o f the f i r s t two ensymes in the catabolic pathway for leucine in liv er and muscle of control and protein-free fed rats. It may be worth noticing that in Neale 's e a r lie r work (1974) when no reduction in oxidative catabolism was observed in rats fed the protein -free d iet: l ) plateau spec ific radioactivity o f 12 output did reach a plateau. Moreover, these animals were not fasted overnight, as they had been in his previous experiments. Pasting w ill tend to obscure the adaptation to low protein diets by increasing the oxidation o f the branched-chain amino acids.It seems, therefore, that the discrepancy between the present results and those of Neale can be resolved i f attention is paid to the deta ils o f the experimental design.r i i i ) Oxidation Rates In VivoSeveral workers have given a single does o f 14C amino acid in vivo, and expressed the rate of oxidation as the proportion of the dose excreted as ,4C02 in the next few hours (MeParlane k Von Holt, 1969 a| Brookes, Owens k Oarriana, 1972» Neale, 1971, 1972). This approach was used in the f iru t part o f this work, since it does give useful qualitative information. However, itpossil127value found by Oar lick , Mi 11ward ft Janas (1973) fo r the average SR of free tyrosine in tissues compared with that in plasma.Thus the use of 1 - 14C leucine in a constant infusion with calcu lation based on the SR in muscle, probably o ffe rs the most accurate method ava ilab le o f measuring the overall oxidation rate o f leucine.As shown in Table 10, a protein -free d iet led to decreased oxidation o f leucine in the rat, thus refuting Neale 's claim (1971, 1972, 1974) that the rat is incapable o f reducing the oxidation rate o f BCAs on a low protein d iet.The equation given above may be written in another way I - the total rata o f oxidation i ■ -0 where yv is the proportiono f dose excreted at plateau and Q is the tota l rate o f amino acid turnover or flux . (Q “ JJateau SR* ( Naterlow ft Stephen,1967).From thia i t follows that a reduction in the absolute rate o f oxidation could be brought about in two ways l —(1 ) By a f a l l in j r , i .o . in the proportion o f the flux which is oxidised.(2 ) By a f a l l in flux , the proportion oxidised reoutining or both fa cto rs could be a ltered .The decrease in oxidation o f leucine is one step towards the reduction o f N-excretion which is known to occur in a low protein d iet (Vaterlow, 1968). Picou ft Taylor-Rberta (1969) have shown that tie f a l l in N—excretion is due to a decrease in the proportion of the flux which is oxidised and excreted and not to a decrease in the overall flux . The present work confirms this (Table 13),128but only when flux rates are determined from the SR o f the intracellu lar free amino acid. Recently, Garlick et n l (1973) found that tyrosine flux was re la t iv e ly unchanged in rata fed a protein-free diet for 3 days. However, by day 21 on the diet, the flux was greatly reduced. In the present experiment, leucine flux remained unaltered by day 7 o f protein-free feeding. In contrast, the interpretation leading from measurements o f the SR plasma amino acid is that both flu x and the proportion o f flux oxidised are reduced.I.( i n ) Oxidation Rates in the Hind-LimbMeasurements o f leucine oxidation rates in perfused hind-limbs o f rats fed a control or protein-free diet confirmed the results obtained both in vi vo and in vi tro . As a result o f the in vitroexperiments carried out ea rlier i t was hypothesised that skeletal muscle was the major s ite of leucine oxidation in the whole animal and that adaptation to d ietary protein intake was most significant in this tissue. However, the results with the perfused hind-limb fa i l to demonstrate this with any degree o f certainty because of the d iff icu lt ie s encountered with the preparation. Vithout further work it was not possib le to improve the estimates in the time ava ilab le .Preliminary work showed that in the perfused hind-limb with constant infusion of a tracer dose o f [ l ^ c j -leucine, tho SR o f free leucine in the perfusate rose to a plateau value. This confirsmd the predictions based on a mathematical su>del drawn up by P.J. Garlick, and described in Section I I IB (i v ) . l%irthemore, i t was predicted that the SR of free leucine in the gastrocnemius ICP pool should follow quite closely the r ise to plateau in thsperfusate. Unfortunately, as may be observed in fig . 12,Section II IC ( v ) , the output o f 14C02 hB<1 not reached plateau at a time when both the SR of free leucine in the perfusate and ICR were predicted to have done so for reasons discussed in Section II IC ( v ). furthermore, as discussed earlier, the preparation was not hiaintained much beyond this period although the 14CC>2 output would be expected to reach plateau by 3 hr. of a constant infusion. Therefore, the estimates of total body muscle leucine oxidation and hind-limb oxidation rates are underestimates. Nevertheless, this method offers the most accurate approach available to determine muscle's contribution to tdtal body leucine oxidation. The calculated leucine oxidation rate in total body skeletal muscle was 7 . 34/Jmoles/hr ia iSOg rat or approximately 25^ o f total body tissue leucine oxidation. In preliminary experiments on perfused livers where plateau **C02 ou-oxo-isovaleric acid dehydrogenase a c t iv it ie s .i?7TABLE 10. Abso lute oxidation rates o f l [ i ^ c] leucinet>ace65TABLE 11. M uscle protein synthesis in vivo. E ffeot o fp ro te in -free feeding. 69TABLE 12. L iv e r protein synthesis rates In vivo - E ffecto f protein-free feeding. 70TABLE 13. F lu x rates of leucine in the whole rat determinedA . from the sp ec ific radioactivity o f free le u c in e in plasma.B . from the average fo r each group o f rate o f th e spec ific rad ioactiv ity o f free leucine in gastrocnemius ICF. 71»TABLE 14. S p e c i f ic radioactivity o f free leucine in plasma(S p ) and gastrocnemius ICF (S i ) in rats fed e i t h e r a 1096 MDpiE o r 0% NDp:K diet fo r one week and the ratio o f Si/Sp in vivo. 76TABLE 15. Concentration o f amino acid mixtures fo r hind-lim b perfusions. 66TABLE 16. P ro p e r t ie s of the perfused hind-limb preparation. 87TABLE 17. R ates o f leucine oxidation In the perfused hind-lim bs e ffe c t of p ro te in -free feeding and variation in th e amino aoid concentrations o f the perfusate. 96TABLE 16. Concentrations of fre e leucine in perfusate andin t r a c e l lu la r flu id o f gastrocnemius muscle a fte r 2 h o u rs perfusion o f the hind-limb, and In plassa9 7TABLE 19.TABLE 20.TABLE 21.TABLE 22.Protein synthesis rates in tho hind-limb: effect o f p rote in -free feeding and variation in the amino acid concentration of the perfusate.Leucine flux rates in the hind-limb preparation: (A ) estimated from SR of free leucine in »plasma* and 1*C02 output and (B ) estimated from SR o f free leucine in gastrocnemius muscle and 1^ C02 output.Leucine flux rates in tota l body muscle estimated on the assumption that skeletal muscle mass is 45% o f tota l body weight.Comparison o f protein synthesis rates in skeletal muscle estimated (A ) in vivo: (B) in the gastrocnemius ousels o f the perfused hind-limb and (C ) from flux rates in the hind-limb.138PAGE9f100102IO**frJBT OP SCHEMESSCHEME X. Reactiono invo lving the radioactivitylabelled carboxyl group in l [ i 1* c ] -1*»0REFERENCESADI BI, S .A ., (1975) Am. J . P h y s i j l . 221. , 829ADIBI, S .A ., PETERSON, J .A . , 4: KRZYSIK, B.A., (1975) Am. J. Physiol 228. 432AGUILAR, T.S ., HARPER, A .E . , A BENEVENGA, N.J. (1972) J. Nutr.102. 1199.AX I , X., OGAVA, K., A JCHJHARA, A. (1968) Biochim, Biophys. Acts.±59, 276.AKI, X., I0K0J1KA, A ., ft ICHIHARA, A. (1969) J. Biochem. 65, 539. ALLEINE, G.A.O., ft PICOU, 0 .1 . (1971) Br. J. Hosp. Med. p.618.AUB, M., ft VATERLOV, J.C . (19 7 0 ) J. Theor.Biol. 26, 243.BEATTY, C.H., CURTIS, S. , YOUNG, M.X., ft BOCHC, R.M. (1974) Am. J.Physiol. 227, 268.BEINERT, H. , (i960) in "The Enzymes", (P.D. Boyer, H Lardy ft K. Myrback, e d i ) , Vol.2, chap.10, Academic Press,New York and London.BLOXAM, D.L., (1971 ) Br. J . N u tr . 26, 393.BONDEN, J .A ., ft CONNELLY, J . L . , (1968) J. B iol. Ches>. 243. 3526. BRAUNSTEIN, A.E., (1960) in "The Enzymes" (P.D. Boyer, H. Ladry ftK. Myrback, eds. ) , vo l.2 , chap.6, Acadesiic Press, New York and London.BROOKES, I.M., OWENS, P .N ., ft GARRIOUS, U.S.. (1972) J. Nutr. 102. 27. BUSE, M.0 ., ft BUSE, J . , (1967) Diabetes. 16, 753.BUSE, M.G., BIGOERS, J .P ., DRIER, C ., ft BUSE, J .P . (1973) J. Biol.Chesi. 248 . 697.BUSE, N.O., BIGGERS, J .P ., PRIDERICI, K.H., ft BUSE, J .P ., (1972)J . Biol .Che*. 24£, 8085.1*1BUSE, M.O., JURSIN1C, S ., * REID, S .S ., (1975) Biochan. J. ¿48, 363.BUSE, M.O., * REID, S .S ., (1975) J. C lin .In raa t. 56, 1250.CAHILL, G.F. Jr. (1971) Diabataa, 20, 785.CLARKE, E .V ., (1957) J . Phyaiol, 136. 380.CONNELI.T, J L ., DANNER, D .J ., A BOOTE», J .A ., (1968) J. Biol.243. 1198.DANCIS, J . , HUTZLER, J . , A LEVITZ, M, (1961 ) Biochan. Biophya, Acta 52, 60.BANNER, O .J., A BONDEN, J .A ., (1966) Fad. Proc. 747.DANNER, D .J ., DAVIDSON, E .V ., A ELSAS, I I L .J ., (1975) Natura, 2^±, 529.DAVSON, A .O .. HIRD, F .J .R ., A MORTON, D .J ., (1967) Arch. Biochan,Biophya. 122. 426.B.VTN, D .H ., (1970) in "Manmalian Protain Matabolian", (H.N. Munro, ad.)V o l.IV p .523-557. Nav YorkiAcadanic Fraaa. FEATHERSTON, V.R., A HORN, O .V ., (1973) J. Nutr. ¿03, 757.FELIO, P ., OVEN, O.E., VAHREN, J . , A CAHILL, O.F. Jr. (1969) J . C lin . lam a*. ¿8, 584.FELIO, P ., A VAHREN, J . , (1971) J. C 'in .Inaaat. ¿O, 2703.FELLER, D.D., A FEIST, E ., (1962) Biochaai, Biophya, Acta, 62, 40.FERN, E .B ., (1975) Ph.D. Thaaia, Un iraraity o f London.FERN, E .B ., A GARLICK, F .J ., (1973) Biochan. J. 134. 1127- FERN, E .B .. A GARLICK, F .J ., (1974). Biochan. J. ¿42, 413.FULK3, R.M., L I, J .B ., A GOLDBERG, A .L ., (1975). J. B io l. Chan. 2JO, 290.OAN, J .C ., A JEPPAT, H., (1967) Biochan, Biophya, Acta, ¿48, 448.OARLICK■ P .J ., A MARSHALL, I . , (1972) J . Naurochan. ¿2. 577OARLICX. P .J ., MILLVARD, D .J ., A JAMES, V .F .T ., (1974) Biochan, J. ¿¿6, 935. OARLICK, P .J ., MILLVARD, D .J ., JAMES, V .P .T ., A VATERLOV, J .C ., (1975) Biochan, Biophya, Acta. 414, 71.11*2COKI)IMO, H.V., HL'PFNER, M. , MOIILEN'BKCK, P ., * BLUME, K.G. (1967)Biochen, Biophys, Acta. 132. 524. GOLDBERG, A .L., (1972) in "Progress in Muscle Biology", p .89-118 New Tork : Marcel Decker.GOLDBERG, A .L.. A ODESSE!, R., (1972) A «. J. Physiol. 223. 1384.HARPER, A.E., (1965) Can. J. Biochen. 43, 1589.KERLONG, P.H., VEÎGAND, D.A., A BUSE, M.O., (1974). Fed. Proc. Fed.Aai. Soc. Exp. B io l. 22*HOLT, L .E ., SNTDEHMAN, S.E ., NORTON, P.M ., ROITMAN, E ., A PINCH, J ., (1963) Lancet 2, 1343.ICHIHARA, A ., A KOTAMA, E ., (1966) J. Biochea, (Tokyo). 59, 160. ICHIHARA, A., NODA, C ., A OOAVA, K. (1973) in "Advances in biayaeRegulation " Voi.11 p.155 (O. Vebcr, ed .) New Tork « M Dekker Ine.ITTERTAH, T.B., PERI ERA, S.M., A DUMM, M.K., (1965), An. J. C lin . Nutr.I L " •JAMES, V .P.T., (1972), Proc. Nutr. Soc. 21* 2 n ‘JAMES, V .P.T., OARLICK, P .J ., SENDER, P.M. A VATERLOV, J .C ., (1976),Clia . S c i. A Noi. Med. ¿O, 523.JAMES, V.P.T., SENDER, P.M., GARLICK, A VATERLOV, J.C ., (1974) in"Dynastic Studies with Radioiaotopea in Medicine V o l.I . Printed by International Atonic Energy Agency. Vienna, Austria. JEFFERSON, L .8 ., KOEHLER, J.O ., A MORGAN, H.E., (1972), Proc. Natl.Acad. S c i. USA 62, 816.JOHNSON, V.A., A CONNELL!, J .L ., (1972), Biocheaiatry, JJ., 1967.JOHNSON, P ., HERRING, B. , A FIELD, J .B ., (1961) Metabolica JO, 415.REFERENCESKAIHARA, S., & VAGNER, H.N. (1968) J. Lab. C lin . Med. J ± , 400 KAPLAN, J.H., & PITOT, H.C. (1970 ) in "Mammalian Protein Metabolism"V o l . IV pp.587-443 (H .N . Munro, ed ).Now York and London : /cademic Press.KREBS, H.A. ( 1972) in "Advances in Bizyme Regulation" Voi. 10, p. 387.(G Weber, ed.) New Tork s M Dekker Ino.LOWENSTEIN, J.M. (1972) P h y s io l . Rev. ¿2, 382.MoFARLANE, I.G . * VON HOW, C . (1969 a) Biochem. J . 111, 5 5 7 .MoFARLANE, I.G . & VON HOW, C . (1969 b) Biochem. J . JJJ., 56 5.MACKENZIE, D.Y. & WOOLF, L . I . (1959) Brit. Med. J . 2.t 90.McLaren, D.S., KAMEL, V .V ., A AYY0ÜB, N. ( 1965) . Am. J. C lin . Nutr. 21* 152 . MAHLER, R.J., SZABO, 0 ., A PEPJHOS, J.C. (1968), Diabetes, 21» 1*MALLETTE, L.E., EXTON, J.H. A PARK, C.R. (1969) J . B io l. Chem. 5713.MANCHESTER, K.J. (1965) B iochea . Biophys. Acta. 100. 295.MANCHESTER, K.L. (1970) Biochem. J. 221* 457.MEIKLE, A.V., & KLAIN, G.J. (1 9 7 2 ) Am. J. Physiol. 222. 1246.KEISTER, A., (1952) J . B io l-

Skeletal muscle metabolism in hypokinetic rats

Abstract

Extracted data

Skeletal muscle metabolism in hypokinetic rats

Abstract

Extracted data

Related items

Related items