Influence of amino acid availability on protein turnover in perfused skeletal muscle

351

Biochimica et Biophysica Acta, 544 (1978) 351--359 © Elsevier/North:Holland Biomedical Press

BBA 28758 INFLUENCE OF AMINO ACID AVAILABILITY ON PROTEIN TURNOVER IN PERFUSED SKELETAL MUSCLE

JEANNE B. LI and LEONARD S. JEFFERSON Department of Physiology, College of Medicine, The Pennsylvania State University, Hershey, Pa. 1 7033 (U.S.A.)

(Received March 17th, 1978)

Summary The effects of amino acids on protein turnover in skeletal muscle were determined in the perfused rat hemicorpus prepration. Perfusion of preparations from fasted young rats (81 _+2 g) with medium containing either a complete mixture of amino acids at five times (5X) their normal plasma levels, a mixture of leucine, isoleucine, and valine at 5X or 10X levels, or leucine alone (10X) resulted in a 25--50% increase in muscle protein synthesis and a 30% decrease in protein degradation compared to fasted controls perfused in the absence of exogenously added amino acids. When the branched-chain amino acids were omitted from the complete mixture, the remaining amino acids (5X) had no effect on protein turnover. The complete mixture at 1X levels was also ineffective. Comparison of the effects of amino acids with those of glucose and palmitare indicated that amino acids were not acting by providing substrates for energy metabolism. The stimulatory effect of amino acids on protein synthesis was associated with a facilitated rate of peptide-chain initiation as evidenced by a relative decrease in the level of ribosomal subunits. This response was not as great as that produced by insulin, and the amino acids did n o t augment the effect of insulin. Although protein synthesis in preparations from fed young rats (130 + 3 g) was stimulated by the addition of a mixture of the branched-chain amino acids (5X) to about the same extent as that observed in the fasted young rats, protein degradation was not affected. Furthermore, neither synthesis nor degradation were affected in preparations from fasted older rats (203 + 9 g) suggesting that the age and or nitritional state of the animal may influence the response of skeletal muscle to altered amino acid levels.

Introduction

The availability of free amino acids appears to play an important role in the control of protein turnover in a number of tissues. Studies with the perfused

352 rat liver indicated that protein synthesis could be stimulated and protein degradation inhibited by raising perfusate amino acid levels above those normally found in rat plasma [1--3]. The effect on hepatic protein synthesis required a mixture of at least 11 amino acids [1], while an effect on degradation was observed with a mixture of four amino acids [3]. Protein synthesis in the perfused rat heart was also more rapid when amino acid concentrations above those normally found in plasma were added to the perfusate [4,5]. The addition of a mixture of the three branched-chain amino acids, or the provision of fatty substrates, was as effective as the complete amino acid mixture at stimulating protein synthesis in perfused heart [6]. Since the heart has the capacity to oxidize branched-chain amino acids [7], it was suggested that the amino acid effect on cardiac protein synthesis was due to increased availability of an oxidizable substrate [6]. Amino acids increased protein Synthesis and inhibited protein degradation in isolated rat diaphragms and the branched-chain amino acids were responsible for these effects [8--10]. One study suggested that all three branched-chain amino acids were effective in regulating protein turnover in diaphragm [8], while another indicated that the effect was specific for leucine [10]. In contrast to heart, diaphragm, which also has the capacity to oxidize branched-chain amino acids [7], did n o t exhibit changes in protein turnover when fatty substrates were added to the incubation medium [8--10]. Results of studies with heart and diaphragm may n o t be indicative of the effects of amino acids on protein turnover in the bulk of muscle tissue in the body. Skeletal muscle, which makes up 45% of the body w e i g h t [11], would have the largest influence on overall nitrogen metabolism in the body. Therefore, we have investigated the effects of amino acids on protein turnover in skeletal muscle using the isolated perfused hemicorpus [12,13]. This preparation permitted simultaneous measurements of protein synthesis, protein degradation, and net amino acid release. Determinations of the levels of ribosomal subunits were made in the same preparations as the measurements of protein turnover and served to identify the site of action of the amino acids in the prorein synthetic pathway. Effects of amino acids were assessed in the presence of insulin, and in preparations from fed and fasted animals. Effects of alternative oxidizable substrates on protein turnover were also determined. Methods

Animals. Male Sprague-Dawley rats were obtained from the Charles River Breeding Laboratories (Wilmington, Mass.). They were provided Wayne Lab Blox or Purina Chow and water ad libitum and maintained on a 12 h light/12 h dark cycle for at least 1 week prior to use. In most experiments, rats weighing an average of 112 _+3 g were placed in individual cages with free access to water and fasted for 48 h. At the time of perfusion, these rats weighed 81 _+2 g. In one experimental series, rats with an initial body weight of 260 ± 8 g were fasted for 72 h as described above; these rats weighed 203 ± 9 g at the time of perfusion. In another experimental series, fed rats weighing 130 + 3 g were used. Hemicorpus perfusion. Details of the perfusion technique are described in previous reports [12,13]. The basic perfusate consisted of Krebs-Henseleit

353 bicarbonate buffer, 3% bovine serum albumin (Pentex fraction V, Miles Laboratories), and sufficient washed bovine erythrocytes to give a hematocrit of 25%. F a t t y acid-free albumin (Sigma), cycloheximide (Sigma), bovine insulin (a gift from Eli Lily Co.), and L-[U-14C]phenylalanine (New England Nuclear) were added to the perfusate as indicated in the table legends. Amino acids were added at normal rat plasma concentrations [13], or at five or ten times those amounts. The first 25 ml of persufate to pass through the preparation were discarded. Then, 75 or 100 ml of persufate were recirculated at a flow rate of 7 or 14 ml/min as indicated in the table legends. Determination of rates of protein synthesis and degradation. Protein turnover measurements were made between 1 and 3 h of perfusion as described previously [13]. Protein synthesis in the gastrocnemius muscle was determined by the incorporation of phenylalanine into protein when the perfusate contained 0.4 mM L-[14C]phenylalanine at a specific activity of 125 mCi/mol. As described previously [13], phenylalanine is a suitable marker for determining protein turnover, since it is neither synthesized nor degraded by this preparation. Furthermore, these concentrations of phenylalanine did n o t alter rates of protein turnover (unpublished observations) and ensured that the specific activity of phenylalanyl-tRNA, the immediate precursor of protein synthesis, was the same as that of the extracellular and intracellular pools of free phenylalanine [14]. The rate of protein synthesis, expressed as nmol phenylalanine incorporated/h per g muscle was calculated by dividing the incorporation of [14C]phenylalanine into muscle protein by the intracellular specific activity of phenylalanine. Protein degradation was measured either b y dilution of [14 C]phenylalanine specific activity or by the release of phenylalanine in the presence of 100 gM cycloheximide [13]. Cycloheximide inhibited protein synthesis by more than 95%, thus minimizing the reutilization of released phenylalanine. Rates of degradation are presented as nmol phenylalanine released/h per g hemicorpus. Perfusate and tissue analyses. The acid-soluble phenylalanine content and radioactivity were determined in extracts of perfusate and frozen gastrocnemius muscle b y fluorimetry [ 13,15 ] and by liquid scintillation spectrophotometry [13], respectively. Incorporation of radioactivity into protein was determined as described previously [5,13]. Ribosomal subunits in homogenates of psoas muscle were separated by sucrose density centrifugation [6] and the RNA content of 60-S and 40-S fractions was measured by alkaline hydrolysis [16]. Statistical analyses. Results are presented as the means _+S.E. The significance of the differences between the means was determined by the two-tailed Student's t-test. Results

48 h fasted small (81 -+ 2 g) and large (203 + 9 g) rats were used to provide animals comparable to those used in similar studies on diaphragm [8] and heart [6], respectively, while the use of fed animals (130 _+3 g) permitted a determination of whether the observed effects of amino acids were restricted to the fastin~ state. Data obtained from measurements of protein turnover in skeletal

4 2 -+ 2 49 ± 4 60-+ 7 " 53 -+ 4 * * * 3 5 -+ 4 6 0 -+ 5 * 60 + 5 " 23 + 3 24-+ 3 21 + 2 30 -+ 4 3 5 +- 9

65 ± 2 1 0 2 -+ 7 *

No addition + 1X a m i n o a c i d s + 5X a m i n o a c i d s + 5X L e u , Ile, V a l

+ 5X a m i n o a c i d s w i t h o u t L e u , Ile, V a l + 10X L e u , Ile, V a l

+ 10X Leu

No addition + 1.5mMpalmitate + 15 glucose

No addition + 5)< Leu, Ile, Val

No addition + 5X L e u , Ile, V a l

Fasted small rats, 81 ± 2 g; p e r f u s a t e contained fraction V

Fasted small rats, 81 + 2 g; p e r f u s a t e contained fat-free a l b u m i n

F a s t e d l a r g e rats, 2 0 3 ± 9 g; p e r f u s a t e contained fraction V

F e d small rats, 1 3 0 + 3 g; p e r f u s a t e contained fraction V

(A)

(B)

(C)

(D)

*=P<0.005. **=P< 0.02. * * * = P < 0 . 0 5 ; all v e r s u s t h e c o n t r o l , n o a d d i t i o n .

Protein synthesis

Additions to perfusate

ExperLmental conditions

p e r i m e n t a l g r o u p s p e r f u s e d at t h e s a m e t i m e .

+ 11 -+ 22 + 8"* + 10 **

8 **

130-+ 5 1 1 4 +- 13

1 1 3 -+ 13 1 0 9 -+ 12

1 7 5 -+ 14 170-+ 5 1 7 8 + 10

125-+

1 8 1 ± 13 1 5 2 +- 13 * * *

191 183 126 127

--Cycloheximide

132-+ 5

134-+ 8 **

1 2 8 +- 6 *

122± 8" 128 + 6 *

172 ± 6

+ Cycloheximide

Protein degradation (nmol phenylalanine]h per g tissue)

7"

67-+ 6 2 8 -+ 5 *

74 ± 7 57 ± 17

131 ± 7 1 3 7 ± 12 125 ± 8

61 +

1 3 2 -+ 11 67 -+ 6 *

130 + 8 1 0 5 ± 21 87± 12"* 6 9 ± 12 *

Net release

H e m i c o r p u s p r e p a r a t i o n s f r o m e i t h e r f e d r a t s , 1 1 2 g r a t s f a s t e d f o r 4 8 h, o r 2 6 0 g r a t s f a s t e d f o r 72 h w e r e p e r f u s e d as d e s c r i b e d i n M e t h o d s . A d d i t i o n s t o t h e b a s i c m e d i u m are i n d i c a t e d in t h e t a b l e . E i t h e r f r a c t i o n V b o v i n e s e r u m a l b u m i n ( f r a c t i o n V ) o r f a t t y a c i d - f r e e a l b u m i n ( f a t - f r e e a l b u m i n ) w a s u s e d i n p r e p a r i n g t h e b a s i c m e d i u m . A m i n o a c i d s w e r e a d d e d a t n o r m a l p l a s m a levels ( 1 X ) o r f i v e a n d t e n t i m e s t h o s e c o n c e n t r a t i o n s (SX a n d 10X). P a l m i t a t e w a s a d d e d as a n a l b u m i n c o m p l e x [ 2 8 ] . P r o t e i n d e g r a d a t i o n w a s m e a s u r e d i n t h e a b s e n c e (--) o r p r e s e n c e ( + ) o f c y c l o h e x i m i d e ( i - 10 -4 M) as d e s c r i b e d p r e v i o u s l y [ 1 3 ] . " N e t r e l e a s e " o f phenylalanine, determined from changes in phenylalanine content of perfusate and tissue [13] in preparations perfused in the absence of cycloheximide, repres e n t e d t h e b a l a n c e b e t w e e n r a t e s o f p r o t e i n s y n t h e s i s a n d d e g r a d a t i o n . F o r p e r f u s i o n o f p r e p a r a t i o n s f r o m l a r g e r r a t s , t h e w a s h o u t v o l u n l e o f perf-usate w a s i n c r e a s e d f r o m 2 5 t o 50 m l , t h e r e c i r e u l a t i n g v o l u m e f r o m 75 t o I 0 0 m l , a n d t h e f l o w r a t e f r o m 7 t o 14 m l / m i n t o c o m p e n s a t e f o r t h e l a r g e r size o f t h e h e m i c o r p u s preparation. In each group of experiments preparations were perfused with medium containing no additions to establish basal (control) rates of protein turnover. E a c h v a l u e p r e s e n t e d is t h e m e a n -+ S.E. o f 8 - - 3 0 d e t e r m i n a t i o n s , e a c h i n v o l v i n g a s e p a r a t e p e r f u s i o n e x p e r i m e n t . S i g n i f i c a n c e w a s t e s t e d b e t w e e n c o n t r o l a n d e x -

EFFECTS OF AMINO ACIDS, PALMITATE, AND GLUCOSE ON SKELETAL MUSCLE PROTEIN TURNOVER

TABLE I

c.n

355 muscle of these different experimental groups are presented in Table I. In the first experimental series (Table I, A), it can be seen that the addition of exogenous amino acids at normal rat plasma levels to the perfusate had no effect on the rate of protein synthesis. Raising the amino acid concentrations to 5X normal, however, increased protein synthesis by 43%, an effect which was mimicked by the addition of a mixture of the branched-chain amino acids (leucine, isoleucine, and valine) at 5X or 10X, or b y the addition of leucine alone at 10X. When the branched-chain amino acids were omitted from the complete mixture, the remaining amino acids (5X) had no effect on protein synthesis. Thus, in agreement with previous studies on diaphragm [8--10], leucine appeared to be responsible for the stimulation of synthesis. Rates of protein degradation were also measured in this experimental series. At 5X or 10X concentrations, amino acid mixtures containing the branched-chain amino acids inhibited protein degradation by approx. 30%, regardless of whether the determinations were made in the absence or presence of cycloheximide. The addition of leucine alone (10X) produced an inhibition of a similar magnitude, suggesting that this amino acid was solely responsible for the effect on degradation. Net phenylalanine release, a reflection of net protein turnover, was reduced b y more than 50% as a result of amino acids stimulating synthesis and inhibiting degradation. Results obtained in diaphragm and heart suggested that protein turnover was affected b y glucose [8], ketone bodies [6], free fatty acids [6] and other oxidizable substrates [6]. Furthermore, previous studies demonstrated that these muscle preparations were capable of oxidizing the branched-chain amino acids [7,20]. Therefore, the second experimental series (Table I, B) investigated the possibility that the amino acid effects noted above were due to the provision of oxidizable substrates. In this series the fraction V albumin used in the perfusion medium was replaced with fat-free albumin, since the fraction V preparation was known to contain fatty acids and other substances [21,22]. In the presence of fat-free albumin, the basal rate of protein synthesis was reduced by 45%, suggesting that the fraction V preparation contained a contaminant which enhanced protein synthesis or that a contaminant of the fat-free albumin preparation inhibited protein synthesis. Neither possibility was investigated further in the present study. These experiments demonstrated that provision of 1.5 mM palmitate b o u n d to the fat-free albumin caused no change in the basal rate of either synthesis or degradation. Similarly, the basal rates of synthesis and degradation were unaffected b y the addition of glucose. The third experimental series (Table I, C) studied the effects of a mixture of the branched~chain amino acids on protein turnover in skeletal muscle of fasted larger rats (203 + 9 g), a size of animal comparable to that used in previous studies on perfused heart [6]. The basal rates for both synthesis and degradation were lower in this series, due to the effects of increasing age on these parameters (ref. 20, and unpublished data). In contrast to the results obtained in fasted y o u n g rats, neither synthesis nor degradation were significantly altered by the addition of the amino acid mixture. The fourth experimental series (Table I, D) investigated the possibility that protein turnover in skeletal muscle of fed rats may respond to amino acids in a manner different from that observed in fasted animals. These rats were compar-

356 TABLE

II

THE EFFECT AND LEVELS

OF BRANCHED-CHAIN AMINO OF RIBOSOMAL SUBUNITS

ACIDS

AND

INSULIN

ON PROTEIN

SYNTHESIS

S m a l l , 1 1 2 g r a t s w e r e f a s t e d f o r 4 8 h a n d p e r f u s e d a s d e s c r i b e d i n M e t h o d s a n d i n T a b l e I. A m i n o a c i d s w e r e p r e s e n t at five o r t e n t i m e s n o r m a l p l a s m a l e v e l s . I n s u l i n w a s p r e s e n t a t 2 5 m u n i t s / m l . P r o t e i n s y n thesis was measured in gastrocnemins muscle between 1 and 3 h of perfusion. Ribosomal subunits were isolated from psoas muscle after 3 h of perfusion. The means ± S.E. of six or more determinations are presented. Conditions

Fed, unperfused Fasted, unperfused 'Fasted, perfused +Insulin +Leu, Ile, Val +Leu +Insulin, Leu, Ile, Val a=p~ b=P~ e = p ,~ d = not

Protein synthesis (nmol phenylalanine/h per g muscle)

48 82 66 72 84

-+ 3 -+ 3 ± 6 + 6 ± 6

a b b a

Ribosomal

subunits

60 S 47 158 145 59 114 111

40 S + 6 ± 18 ± 4 + 2 a,d -+ 7 a +- 7 a

33 88 93 35 79 76

• 3 ± 10 • 3 • 2 a,d • 4 b +_ 8 c

0.001. 0.01. 0 . 0 2 vs. f a s t e d p e r f u s e d . different from fed, unperfused.

able in age and initial weight to the animals used in the first series (Table I, A). The fed rats exhibited higher basal rates of synthesis and lower basal rates of degradation than the fasted ones (compare series D with A). It can be seen that muscle protein synthesis in the fed rats was stimulated by the addition of a mixture of the branched-chain amino acids, to about the same extent as that observed in fasted animals. In contrast, protein degradation in the fed rats was not affected by the amino acid mixture whereas the process was significantly inhibited under similar conditions in the fasted animals. In an a t t e m p t to identify the step in protein synthesis which was affected by amino acids, ribosomal subunit levels in psoas muscles of fed and fasted rats were measured before and after perfusion under the conditions used in experimental series A above. As previously shown [19,21], fasting produced a block in peptide-chain initiation in skeletal muscle which was manifested by a 2-fold increase in ribosomal subunit levels (Table II). In fasted animals, these levels were n o t further increased during control perfusions, but were restored to the fed state in association with a stimulation of protein synthesis following perfusion in the presence of insulin. These results confirmed previous work showing that insulin stimulated peptide-chain initiation in skeletal muscle [13]. The data in Table II also show that the stimulated rate of protein synthesis, which was produced by the addition of the branched-chain amino acids or leucine alone, was likewise associated with a reduction in ribosomal subunit levels. This finding suggested that peptide-chain initiation was the step in the protein synthetic pathway which was stimulated by amino acids. This suggestion was supported by the finding that amino acids, in the presence of insulin, produced no further stimulation of protein synthesis.

357 Discussion These experiments define the conditions required for demonstrating an effect of amino acids on protein turnover in perfused skeletal muscle. In fasted young rats, skeletal muscle responds to the increased availability of amino acids in much the same manner as perfused heart and incubated diaphragm. A complete mixture of amino acids at five times normal plasma levels caused a stimulation of protein synthesis and an inhibition of protein degradation in skeletal muscle, while lower amino acid concentrations were required to produce these effects in heart and diaphragm [6,8]. A mixture of leucine, isoleucine and valine were as effective as the complete mixture at stimulating synthesis and inhibiting degradation in skeletal muscle, which agrees with findings in heart [7] and diaphragm [8,10], b u t differs with findings in perfused liver in that other amino acids were required to produce effects on protein turnover [1,3]. Of the three branched-chain amino acids, leucine alone could account for the observed effects in skeletal muscle and in diaphragms from normal rats [8--10], whereas isoleucine and/or valine were also effective in diaphragms from hypophysectomized [8] or diabetic [9] rats, and in hearts from normal rats [6]. The stimulatory effect of amino acids on protein synthesis was manifested in perfused skeletal muscle of fed y o u n g rats, b u t n o t in muscle of fasted older rats. Moreover the inhibitory effect of amino acids on protein degradation was not observed in muscle of either fed young rats or fasted older rats. The explanation for these differing effects is n o t apparent. Comparison of incubated diaphragms from fasted young rats with those from fed animals showed that the amino acid effect on protein synthesis was greater after fasting, while the effect on degradation was the same regardless of whether the diaphragms were from fasted or fed rats [8]. In addition, provision of amino acids altered both protein synthesis and degradation in perfused hearts taken from fasted older animals comparable to the ones used in the present experiments [6,22]. As discussed below, the mechanism by which amino acids affect protein synthesis might involve a metabolic intermediate, the intracellular concentration of which may vary depending on the age and/or nutritional state of the animal or the type of muscle tissue investigated. It is not presently known, though, h o w branched-chain amino acids affect protein degradation, although this study and others [9,22] suggest that the effect is mediated by leucine or a metabolic product of leucine. There is uniform agreement regarding the site in the protein synthetic pathway that is influenced by amino acid availability. It was suggested on the basis of studies in perfused liver that limited amino acid availability resulted in development of a block in peptide-chain initiation [2] and that formation of the block was prevented by the addition of five times normal plasma levels of all amino acids to the perfusion medium [2]. The stimulatory effect of the branched-chain amino acids on protein synthesis in perfused heart was also associated with a facilitated rate of initiation [6]. The present experiments demonstrate that the stimulation of protein synthesis in skeletal muscle by branched-chain amino acids or leucine alone was also associated with a facilitated rate of initiation [6]. When compared to insulin, which also acts on initiation [ 13], the branched-chain amino acids or leucine alone were only partially

358 effective in restoring ribosomal subunits to the levels found in muscle of normal fed animals. In contrast to the abstract of Atwell et al. [23], which suggests that the amino acid effect on initiation was additive or permissive with that of insulin, the present study revealed no effect of the branched-chain amino acids when they were added in combination with the hormone. The results shown here agree with those from studies on perfused heart indicating that in the presence of insulin the branched-chain amino acids had no effect on protein synthesis or degradation [24]. T w o potential mechanism can be advanced to explain the stimulatory effect of branched-chain amino acids on initiation. First, several lines of evidence suggest that a block in initiation may be mediated by the accumulation of deacylated t R N A [25--27]. Therefore, a possible explanation for the stimulatory effect of the branched-chain amino acids on initiation in muscle is that the formation of higher levels of their respective aminoacyl-tKNA derivatives reduced the inhibitory influence of deacylated tRNA. Since the effect was relatively specific for leucine in both incubated diaphragm [10] and perfused skeletal muscle, leucyl-tRNA formation may be particularly important in controlling initiation in these tissues. The second possible mechanism for explaining the stimulatory effect of the branched-chain amino acids on initiation in muscle is that their effect is mediated by a metabolic intermediate arising from the oxidation of these compounds. Studies in perfused heart supported this mechannism, since provision of a number of oxidizable substrates produced an effect on initiation similar to that seen with the branched-chain amino acids [22]. In perfused skeletal muscle and incubated diaphragm [8], however, provision of ketone bodies or fatty acids as alternative oxidizable substrates did n o t stimulate protein synthesis, nor did the provision of ~-ketoisocaproate (ref. 10, and unpublished data), the metabolic product of leucine transamination, which is oxidized faster than equimolar concentrations of leucine [10]. Therefore, these studies do n o t support the possibility of an oxidative metabolite of the branched-chain amino acids accounting for their effect on initiation in skeletal muscle. Thus, resolution of the biochemical mechanism of this effect must await further investigation. Acknowledgements This work was supported by grants from the National Institute of Arthritis, Metabolism, and Digestive Diseases, Bethesda, Md. (AM 15658), the Muscular D y s t r o p h y Association, and the American Diabetes Association. L.S.J. is an Established Investigator of the American Diabetes Association. The authors are grateful to Mary E. Burkart and Richard A. Guinivan for expert technical assistance and to Jeanette Schwartz for help in the preparation of this manuscript. References 1 J e f f e r s o n , L.S. and Korner, A. ( 1 9 6 9 ) B i o c h e m . J. 1 1 1 , 7 0 3 - - 7 1 2 2 J e f f e r s o n , L.S;, R o b e r t s o n , J . W . a n d T o l m a n , E.L. ( 1 9 7 2 ) in G r o w t h a n d G r o w t h H o r m o n e (Pecile, A. and Miller, E.E., eds.), p p . 1 0 6 - - 1 2 3 , E x c e r p t a Medica, A m s t e r d a m 3 W o o d i s d e , K . H . and M o r t i m o r e , G . E . ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 6 4 7 4 ~ 6 4 8 1 4 M o r g a n , H . E . , Earl, D . C . N . , B r o a d u s , A., W o l p e r t , E.B., Giger, K . E . and Jefferson, L.S. ( 1 9 7 1 ) J. Biol. Chem. 246, 2152--2162

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