NUTRITION RESEARCH, Vol. 11, pp. 1155-1166,1991 0271-5317/91 $3.00 + .00 Printed in the USA. Copyright (c) 1991 Pergamon Press plc. All rights reserved.

AN IN VIVO EXAMINATION OF THE EFFECTS OF LEUCINE ON SKELETAL MUSCLE PROTEIN SYNTHESIS IN THE FASTING RAT

Gregory J. Wibert, M.S., Donald K. Layman, Ph.D., and Soon O.K. Hong, Ph.D.

Divisions of Nutritional Sciences and Foods and Nutrition, University of Illinois, 274 Bevier Hall, 905 S. Goodwin, Urbana, Illinois 61801

ABSTRACT

Leucine has been shown to stimulate protein synthesis in in vitro muscle preparations. However, in vivo studies have been unable to reproduce these results consistently. The purpose of the current study was to examine the effects of leucine on in vivo protein synthesis using a bolus injection of 14C-tyrosine as a tracer. Intraperitoneal injections of leucine resulted in a linear increase in muscle free leucine up to 6 times control values. Leucine stimulated protein synthesis in skeletal muscles from fasted rats. The extensor digitorum Iongus and plantaris synthesis rates in 1-day fasted rats were increased with leucine in the absence of added insulin, while synthesis rates in the soleus remained unchanged. In fed rats, there was no stimulation of protein synthesis with leucine administration. These results indicate that leucine has the potential to stimulate muscle protein synthesis in vivo, however, the response is dependent on the nutritional status and the muscles selected.

KEY WORDS: Leucine, Protein synthesis, Fasting

INTRODUCTION

Over the past 15 years, the metabolism of the branched-chain amino acids, leucine, isoleucine and valine, and their role in the regulation of protein metabolism have been extensively investigated. In vitro evidence indicates that branched-chain amino acids produce anabolic effects on skeletal muscle protein metabolism by a mechanism which involves more than just their participation as precursors. These anabolic effects have been obtained by adding the branched-chain amino acids together or by adding leucine alone. Absence of leucine eliminates the anabolic effects (1,2). Branched-chain amino acids have been shown to stimulate protein synthesis and to d'ecrease protein degradation in muscles (1-4). In skeletal muscles from normal rats, leucine appears to be the specific amino acid which enhances protein synthesis (4,5). This enhancement is not dependent upon leucine metabolism (3,6) nor is it reproduced by administration of energy substrates such as glucose, fatty acids (7), lactate or ketones (6). Furthermore, in vitro results have shown that

1155

1156 G.J. WlBERT et al.

the leucine effect was most reproducible in muscles from fasted rats (4,5) and was enhanced when muscles were pre-incubated with insulin (2).

Experiments in vivo have been unable to produce the dramatic stimulation of protein synthesis found in vitro. Many catabolic models have been used to examine the stimulatory effect of branched-chain amino acids on protein synthesis in vivo. In studies using sepsis (8), trauma (9), or a post-operative model (10), branched-chain amino acids have been shown to improve nitrogen balance in rats, while other-studies have failed to show any effect when compared to either a saline control (11) or an amino acid control (12). In addition to measuring nitrogen ba.lance, two of these studies measured skeletal muscle protein synthesis and observed no stimulation with added leucine (8,10).

The effects of branched-chain amino acids on skeleta~ muscle protein synthesis in fasting rats have been examined by two laboratories (13-16). Buse (13) found increased ribosomal activity and increased muscle protein synthesis when branched- chain amino acids were infused after insulin. However in subsequent studies, Garlick and co-workers (15) observed no effect of branched-chain amino acids in fasted rats, but found that branched-chain amino acids did increase tissue sensitivity to insulin in post-absorptive rats (16).

The purpose of this study was to examine the effects of leucine on in vivo protein synthesis. In vitro and in vivo comparisons are difficult due to differences in the ,catabolic state of the muscles, differences in specific muscles and fiber types, differences in leucine dosage, and differences in duration of response. The method and experimental conditions selected in this study were designed to minimize the differences between in vitro and in vivo approaches.

MATER. IALS AND METHODS

Animals Male Sprague-Dawley rats, weighing 55-60 g, were purchased from

Harlan/Sprague-Dawley (Indianapolis, IN) and housed individually under conditions controlled for temperature and humidity with a 12-hour light/dark cycle. Animals were fed a commercial pelleted diet (Ralston Purina, St. Louis, MO). Final body weights for rats in all experiments were between 80 and 110g.

Experiments Experiment 1: Time course of changes in 14C-tyrosine concentrations in serum and skeletal muscle after ip injection

For determination of serum and tissue specific activities (cpm/nmole tyrosine) and tyrosine incorporation into muscle (cpm/mg fresh tissue), rats were sacrificed at 10, 30, 45, and 60 minutes. After ip injection of 10 mCi L-[U-14C]-tyrosine and 18mmoles of unlabeled tyrosine, serum and tissue specific activities were measured at each time point. When higher doses of unlabeled tyrosine were administered (24 or 50 mmoles per 100 g body weight), the limited solubility of tyrosine increased individual variation even though specific activities remained relatively constant throughout the time period.

LEUCINE AND PROTEIN SYNTHESIS 1157

Experiment 2: Effects of different doses of leucine on intracellular free leucine and protein synthesis in muscles of fed rats

To determine the effects of leucine on protein synthesis, rats were injected ip with either saline or one of two doses of leucine, 80mmoles or 160 mmoles, 5 minutes prior to the ip injection of the tyrosine label. Protein synthesis rates for the soleus and extensor digitorum Iongus (EDL) muscles were determined using a bolus injection method (17,18) as described below. Animals were sacrificed at 10 or 40 minutes after tyrosine injection (n=5). Free intracellular leucine was determined on muscles isolated ,15 minutes after injection of leucine and 10 minutes after injection of tyrosine to insure saturation of the cellular free leUcine pool.

Experiment 3: Comparison of the effects of leucine on skeletal muscle protein synthesis in fed and fasted rats

A single dose of 160 mmoles of leucine was injected into fed and 1-day fasted rats in the same manner as in Experiment 2. Animals which served as controls were sham injected with saline. Rates of protein synthesis in the soleus, EDL, and plantaris were determined.

Determination of Rates of Protein Synthesis Protein synthesis rates were determined using the bolus injection method

developed by Henshaw (17). The method was modified to accommodate the use of 14C-tyrosine as the tracer. A smaller flooding dose of tyrosine was used (18 mmoles) and protein synthesis was measured for 30 minutes (i.e. from 10 to 40minutes post tyrosine injection).

Animals were injected ip with 1.5 ml of solution containing 10 mCi L-[U-14C]-tyrosine and 18 mmoles of unlabeled tyrosine as defined in Experiment 1. After either 10 or 40 minutes of labeled tyrosine incorporation into protein, animals were sacrificed by decapitation. Muscles were rapidly removed and homogenized in 10% trichloroacetic acid with a Polytron homogenizer (Brinkman Instruments, Westbury, N.Y.). The homogenates were centrifuged and the acid-soluble supernatants were removed. Aliquots of the supernatants were used to determine radioactivity by liquid scintillation counting (Model LS 9000, Beckman Instruments, Palo Alto, CA) and to determine free tyrosine concentration by the fluorometric procedure of Waalkes and Udenfriend (19). For measurement of 14C-tyrosine incorporation into protein, the acid precipitated material was washed with 5% trichloroacetic acid and ethanol- ether (1:1) and then solubilized in 0.5 M NaOH for 20 minutes at 80~ A small aliquot of the solubilized protein was used for determination of protein content (20) and a 2 ml aliquot, added to 10 ml Aquasol (New England Nuclear, Boston, MA), was used to determine radioactivity.

Serum specific activity was determined from trunk blood collected after decapitation. Samples were left to clot at room temperature for 20 minutes and then centrifuged. Serum samples were pooled, deproteinized with 20% trichloroacetic acid, and then centrifuged. Specific activity was determined on the acid-soluble fraction as described for muscle.

Protein synthesis was estimated as the rate of incorporation of 14C-tyros ine into muscle proteins (cpm/mg fresh tissue or protein) for 30 minutes divided by the

1158 G.J. WlBERT et al.

average specific activity of free tyrosine (cpm/nmole tyr) in the 10 and 40 minute samples, i.e.

(Sp t40-Spt l 0)/ (Sit40-Sit l 0)/2

Spt40 = 14C-tyrosine incorporated into muscle protein 40 minutes after tyrosine ip injection.

SPt l0 = the mean 14C-tyrosine incorporated into muscle protein 10 minutes after tyro~ine ip injection (4-5 rats per group).

Sit40 = total tissue free tyrosine specific activity 40 minutes after tyrosine ip injection.

Sit10 = the mean free tyrosine specific activity 10 minutes after tyrosine ip injection (4-5 rats per group).

Cellular Free Leucine Determination Fifteen minutes after injection of leucine, animals were decapitated. Soleus

muscles were removed and frozen in liquid nitrogen until analysis. Muscles from 4 animals in each of the three treatments: control, 80, and 160 mmoles of leucine, were pooled and deproteinized in ice-cold 3.5% sulfasalycilic acid. An aliquot of the supernatant was diluted 1"1 with 0.3 N lithium citrate buffer (pH 2.2) and analyzed in an amino acid analyzer (Beckman 121) using an external standard of aminobutylic acid.

Statistical Analysis Experimental results are presented as means + standard errors. Statistical

difference between means was analyzed by Student's t-test. Two-tailed t-values were used for analysis with a probability less than p = 0.05 taken as significant.

RESULTS

Experiment 1: Time course of changes in 14C-tyrosine concentrations in serum and skeletal muscle after ip injection.

The purpose of Experiment 1 was to validate the use of tyrosine as a tracer in the bolus injection method of Henshaw et al. (17). A smaller flooding dose of tyrosine was used (18 mmoles) and protein synthesis was measured for 30 minutes (i.e. 10- 40 minutes). The time course changes of serum and tissue tyrosine specific activities and of muscle tyrosine incorporation are shown in Figures 1, 2 and 3. Serum specific activity was measured in pooled serum samples from four animals at each time point. Figure 1 shows the changes in plasma specific activity during the 60 minutes after injection of 14C-tyrosine and 18 mmole unlabeled tyrosine. Comparison of serum (Fig. 1) and tissue (Fig. 2) specific activities indicates that serum specific activity remained higher than tissue specific activity throughout the measurement of protein s~/nthesis (i.e., 10-40 minutes). As shown in Figure 3, 14C- tyrosine incorporation into tissue protein increased linearly between 10 and 45 minutes in the soleus, EDL, and plantaris,

LEUCINE AND PROTEIN SYNTHESIS 1159

500

4OO

300 o

E 200

E O .

o

l e d

0 1 ' 0 ' ' ' ' ' " ' 7 " 20 30 40 50 60 0

T i m e ( r a i n ) FIG. 1 Time course of serum specific activity after ip injection of 10 mCi L-[U-14C]-tyrosine and 18 mmoles of unlabeled tyrosine

300

200

O

E e -

100 n

o

B Soleus �9 F_DL [] Plantaris

, . i i i i !

0 10 20 30 40 50 Time (rain)

FIG. 2 Time course of the muscle acid-soluble specific activity after ip injection of 10 mCi L-[U-14C]-tyrosine and 18 mmoles of unlabeled tyrosine

Experiment 2: Effect of different doses of leucine on intracellular free leucine and protein synthesis in muscles of fed rats.

Intraperitoneal administration of leucine produced a linear response in the concentration of muscle free leucine (Fig. 4). Intracellular free leucine was increased in the soleus fifteen minutes after injection. The injection of 80 and 160 mmoles of leucine, equivalent to 1/6 and 1/3 of the daily requirement for leucine (19,21) respectively, increased the tissue free concentration by 4.0-fold and 6.8-fold, respect ively.

The in vivo effects of a single, large dose of injected leucine on skeletal muscle protein synthesis of fed rats were examined. Rates of protein synthesis were determined from 14C-tyrosine incorporation and tyrosine specific activity. Values for 14C-tyrosine incorporation and tyrosine specific activities in fed animals are presented in Table 1. These values were calculated as described in Materials and

1160 G.J. WlBERT et al.

40

3 0

20 ,"" Q Soleus

D. El l_ o lO

�9 Plantaris

0

. . . . 'o ' 'o ' 0 10 20 30 4 50 6 70 Time (min)

FIG. 3 Incoporation of tyrosine into muscle proteins after ip injection of 10 mCi L-[U-14C]-tyrosine and 18 mmotes of unlabeled tyrosine

Methods. Both levels of leucine appeared to increase the incorporation of 14C- tyrosine into muscle proteins. However, concomitant elevation of the specific activity resulted in no change in the rate of protein synthesis. Injection of 160 mmoles of leucine elevated the rates of protein synthesis slightly in the EDL. However, this stimulation was not significant at p < 0.05 in fed rats. Injection of 80 mmoles of leucine did not affect the rates of protein synthesis in either soleus of EDL, (Table 1). The slow-twitch, oxidative soleus muscle had higher synthesis rates than the fast-twitch, glycolytic EDL muscle, which is in accord with previous findings in our lab (4).

o e-

='.=_ U . " "

O

r

FIG. 4

1

control 80 160 Leucine level (umoles)

Intracellular free leucine in the soleus 15 minutes after an ip injection

Experiment 3: Comparison of the effects of leucine on skeletal muscle protein synthesis in fed and fasted rats

The results from an ip injection of 160 mmoles of leucine into fed and 1-day food deprived rats are presented in Table 2. Fractional synthesis rates (FSR) were calculated assuming that muscle protein contained 155 mmoles of tyrosine per gram

LEUCINE AND PROTEIN SYNTHESIS 1161

protein (22). The average FSR was 18.9% per day in the three hind limb muscles from fed rats and 11.7% per day for 1-day fasted rats (Table 2).

The effects of food deprivation on in vivo protein synthesis of white and red muscles can be compared in Table 2. In the muscles with a preponderance of white muscle fibers, EDL and plantaris, protein synthesis was decreased to 50% of fed controls after 24 hours of food deprivation, whereas, protein synthesis in the red soleus was decreased by approximately 20%.

Leucine increased the synthesis rates in the EDL and plantaris muscles of fasted rats by 29% and 24%, respectively. Leucine treatment also increased the rate of incorporation of 14C-tyrosine into protein in the soleus. However, there was no stimulation of the rate of protein synthesis. Leucine did not affect the rate of synthesis in any of the muscles from fed rats (Table 2). This lack of stimulation by leucine is in agreement with the findings from Experiment 2.

TABLE 1

In Vivo Effects of Leucine on Muscle Protein Synthesis of Fed Rats 1

14C Incorporation Tyrosine Treatment 2 into Protein Specific Activity Protein Synthesis

nmol tvr cpm/mg tissue cpm/nmol tyr mg tissue-30 min

Soleus Control 20.6+ 2.3 203+ 22 0.102+ 0.012 (+) 80 Leu 22.8 + 0.8 225 + 24 0.101 + 0.004 (+) 160 Leu 25.6+ 1.3 232+ 28 0.110+ 0.006

EDL Control 17.0+ 2.4 223+ 24 0.076 + 0.011 (+) 80 Leu 18.1 + 1.2 239 + 20 0.076 + 0.005 (+) 160 Leu 21.1 + 1.5 235 + 20 0.089 + 0.007

1 Results are presented as means + SEM with n=4. 2Control: injected with saline; (+) 80 Leu: injected with 80 mmoles of leucine; (+) 160

Leu: injected 160 mmoles of leucine.

DISCUSSION

In vitro skeletal muscle incubations which have examined the effects of leucine on protein synthesis generally have high catabolic rates (4), measure protein synthesis over short time periods using supraphysiological concentrations of leucine (2,4), and require fewer approximations and assumptions in the calculation of protein synthesis rates than in vivo infusion methods (18,23). The in vivo protocol used in these studies simulated these conditions. Young fasted rats were used (100g) because their catabolic rates are higher than adults (24). Intracellular leucine concentrations reached 6 times controls values and protein synthesis was measured during a 30 minute period. A single bolus injection of 14C-tyrosine was used

1162 G.J. WlBERT et al.

allowing us to choose the time of sacrifice at the point where serum and tissue specific activities were similar (comparison of Fig. 1 and 2). This resulted in a homogeneity of specific activities among compartments (i.e. plasma, extracellular and intracellular) at the time of sacrifice and eliminated the need to correct for the extracellular pool specific activity (18, 23). Finally, tissue specific activity was measured at two distinct time points, eliminating the need to achieve a plateau in specific activity instantaneously or the need to approximate its rise to plateau mathematical ly.

TABLE 2

In Vivo Effects of Leucine on Protein Synthesis in Fed and Fasted Rats 1

14C incorporation Tyrosine Treatment 2 into Protein Specific Activity Protein Synthesis FSR3

com nmol tvr mg prot-30 min cpm/nmol tyr mg prot-30 min %/day

FED Soleus

Control 135+ 3 221+ 13 0.613 + 0.013 19.0 (+) 160 Leu 148 + 9 228 + 10 0.649 .+_ 0.055

E)L Control 112+ 5 205+ 8 0.547+ 0.023 16.9 (+) 160 Leu 118+ 10 209+ 5 0.567+ 0.050

Plantar is Control 130+ 8 192+ 8 0.675+ 0.041 20.9 (+) 160 Leu 123 + 11 200.-h 4 0.613 + 0.053

Soleus Control 118• 3 250+ 19 0.476+ 0.011 14.7

(+) 160 Leu 136+ 6 *4 290+ 19" 0.468+ 0.021 EDL

Control 61+ 4 211+ 11 0.291+ 0.018 9.0 (+) 160 Leu 85+ 3 * * 227+ 12 0.376+ 0 .015"*

Plantar is Control 64+ 6 189+ 13 0.337+ 0.003 11.5 (+) 160 Leu 85+ 7" 202+ 7 0.420.+_ 0.031"

1 Data are presented as means + SEM with n=6. 2Control: injected with saline; (+) 160 Leu: injected 160 mmoles of leucine. 3Fractional Synthesis Rate (FSR) is calculated assuming that muscle protein contains 155 mmoles tyrosine/g protein (22). 4Significance by Student's t-test: *p < 0.05, **p < 0.01.

The bolus injection of tracer was adapted from the work of Henshaw et al. (17). In comparison to Henshaw et al., the tracer in Experiment 1, 14C-tyrosine, required a smaller flooding dose of tyrosine (18 mmoles), and the ip injection produced a specific acitivity that was stable for a longer period. Tyrosine has not been used in a

LEUCINE AND PROTEIN SYNTHESIS 1163

single bolus injection method before now due to its limited solubility. However, it is an ideal tracer because it is not metabolized in muscle and produces a stable precursor pool and linear incorporation (Fig. 2 and 3). Tissue free tyrosine specific activities were relatively unchanged between 10 and 40 minutes after 14C-tyrosine injection (Fig. 1).

Fractional synthesis rates (16.9-20.9%/day) in muscles of fed rats (Table2) are in agreement with weight matched FSR reported in the literature from studies utilizing either a~ single injection method (16.5%/day) in the gastrocnemius muscle (15) or a constant infusion technique (16.1-29.0%/day) in the gastrocnemius (24). Thus, the values in this study are consistent with literature values.

In agreement with results from Experiment 2, muscles from fed rats in Experiment 3 did not respond to a high dose of leucine (160 mmoles). The lack of a stimulatory effect of leucine on muscle protein synthesis was not unexpected, since in the fed condition muscle protein synthesis may be at its maximum efficiency and would be unlikely to respond to additional anabolic factors (15). Previous reports showed that injection of insulin into normal fed rats (15,25) or feeding of excess protein above the requirement to growing rats (26) does not stimulate muscle protein synthesis.

With rats deprived of food for 24 hours, leucine stimulated protein synthesis in the EDL and plantaris muscles (Table 2). The stimulatory effect of leucine on synthesis appeared to occur in muscles that were most influenced by the food restriction. The rates of synthesis in the fast-twitch EDL and plantaris muscles were reduced to almost one-half of the normal rate after 24 hours of food deprivation. In contrast, the red soleus muscle, which maintained a relatively normal level of protein synthesis after the 24-hour fast, failed to respond to leucine. The catabolic states which developed in the EDL and plantaris muscles may be necessary for leucine to exert anabolic effects.

Two groups have examined the effects of leucine and/or branched-chain amino acids on the regulation of protein metabolism during fasting conditions. Buse (14) determined synthesis rates with a 6 hr infusion using 100 g rats deprived of food for 48 hours. She reported that branched-chain amino acids stimulated protein synthesis in the soleus but had no effect on the gastrocnemius muscle. The catabolic condition which developed in the soleus muscle in her study should be similar to that in plantaris and EDL muscles of 24-hour starved rats in the present study, since the soleus develops a catabolic condition more slowly than muscles composed of white fibers (4,27). Data from our study and Buse's study suggest that development of a specific catabolic state in muscles of starved animals may be a precondition for leucine to stimulate muscle protein synthesis.

In contrast to our results, Garlick's group (15) reported that 100 mmoles of leucine had no effect on protein synthesis in the gastrocnemius muscle of 2-day starved and 9-day protein deprived rats. McNurlan et al. (15) administered leucine iv and measured synthesis using 14C-phenylalanine plus a large dose (150 mmoles) of unlabeled phenylalanine. Differences between their results and the results of the current study may be explained by the degree of the catabolic conditions in McNurlan's study. After both the 2-day total food deprivation and the 9-day protein deprivation,

1164 G.J. WlBERT et al.

the rates of synthesis in the gastrocnemius muscle were reduced to between 25% and 35% of the fed rate, indicating severe catabolism. Buse (14) showed that under similar catabolic conditions, the gastrocnemius failed to respond to infused branched-chain amino acids. In our study, the protein synthetic rates of the muscles were reduced to 77% (soleus) and 53% (EDL) of fed rates.

The mechanism by which leucine enhanced protein synthesis in vivo is unclear. In rats and dogs, leucine has been shown to increase plasma levels of insulin (15,28). However, human studies have reported that plasma insulin remained unchanged when leucine was infused (29). Buse (14) indicated that leucine's effect on synthesis is more than a stimulation of insulin release. Recent in vivo human studies agree that tissue sensitivity to insulin increased with amino acids infusions (30,31,32). This effect was reproduced in fasting rats by administration of branched-chain amino acids alone (16). The current protocol may be useful in examining the interaction between leucine and insulin in vivo.

In conclusion, in vitro results have indicated a stimulatory effect of leucine on skeletal muscle protein synthesis, however these results have not been confirmed in vivo. Our study has used an in vivo bolus injection approach to simulate in vitro conditions used to study the effects of leucine on skeletal muscle protein synthesis. This protocol could be a useful tool in addressing the conflicts between in vitro and in vivo experimental paradigms. Our results indicated a linear response of tissue free leucine with increasing ip concentration. We showed a stimulatory effect of leucine in vivo in fasted animals without added insulin and also demonstrated a lack of effect in fed animals. The involvement of insulin in the leucine stimulation of protein synthesis can not be ruled out by these studies.

REFERENCES

t. Buse MG, Reid SS. Leucine, a possible regulator of protein turnover in muscle. J Clin Invest 1975; 56:1250-1261.

2. Fulks RM, Li JB, Goldberg AL. Effects of insulin, glucose and amino acids on protein turnover in rat diaphragm. J Biol Chem 1975; 250:290-298.

3. Mitch WE, Clark AS. Specificity of the effects of leucine and its metabolites on protein degradation in skeletal muscle. Biochem J 1984; 222:579-586.

4. Hong S.-O, Layman DK. Effects of leucine on in vitro protein synthesis and degradation in rat skeletal muscles. J Nutr 1984; 114:1204-1212.

5. Hasselgren P-O, James JH, Warner BW, Hummel RP Ill, Fischer JE. Protein synthesis and degradation in skeletal muscle from septic rats. Arch Surg 1988; 123:640-644.

6. Tischter M, Desaultes M, Goldberg AL. Does leucine, leucyl-tRNA or some metabolite of leucine regulate protein synthesis and degradation in skeletal and cardiac muscle. J Biol Chem 1982; 257:1613-1621.

LEUCINE AND PROTEIN SYNTHESIS 1165

7. Li JB, Jefferson LS. Influence of amino acid availability on protein turnover in perfused skeletal muscle. Biochim Biophys Acta 1978; 544:351-359.

8. Hasselgren P-O, LaFrance R, Pedersen P, James JG, Fischer JE. Infusion of a branched-chain amino acid-enriched solution and a-ketoisocaproic acid in septic rats: Effects on nitrogen balance and skeletal muscle protein turnover. J Paren Enteral Nutr 1988; 12:244-249.

9. Freund H, Yeshimura N, Fischer JE. The role of alanine in the nitrogen conserving quality of the branched chain amino acids in the po'st injury state. J Surg Res 1980; 29:23-30.

10. Freund H, Yoshimura N, Lunetta L, Fischer JE. The role of the branched chain amino acids in decreasing muscle catabolism in vivo. Surgery 1978; 83:611-618.

11. Kirvela O, Takala J. Postoperative parenteral nutrition with high supply of branched-chain amino acids: effects on nitrogen balance and liver protein synthesis. J Paren Enteral Nutr 1986; 10:574-577.

12. Blackburn GL, Moldawer LL, Usui S, Bothe Jr. E, O'Keefe SJD, Bistrian BR. Branched chain amino acid administration and metabolism during starvation, injury and infection. Surgery 1979; 86:307-315.

13. Buse MG, Atwill R, Mancusi V. In vivo effect of branched chain amino acids on the ribosomal cycle in muscles of fasted rats. Horm Metab Res 1979; 11:289-292.

14. Buse, MG. In vivo effects of branched chain amino acids on muscle protein synthesis in fasted rats. Horm Metab Res 1981; 13:502-505.

15. McNurlan MA, Fern EB, Garlick PJ. Failure of leucine to stimulate protein synthesis in vivo. Biochem J 1982; 204:831-838.

16. Garlick P J, Grant J. Amino acid infusion increased the sensitivity of muscle protein synthesis in vivo to insulin. Biochem J 1988; 254:579-584.

17. Henshaw EC, Hirsch CA, Morton BE, Hiatt HH. Control of protein synthesis in mammalian tissues through changes in ribosome activity. J Biol Chem 1971; 246:436-446.

18. McNurlan MA, Tomkins AM, Garlick PJ. The effects of starvation on the rate of protein synthesis in rat liver and small intestine. Biochem J 1979; 178:373-379.

19. Waalkes TP, Udenfriend S. A fluorometric method for the estimation of tyrosine in plasma and tissue. J Lab Clin Med 1957; 50:733-736.

20. Lowry OH,, Rosebrough N J, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193:265-275.

1166 G.J. WlBERT et al.

21. NRC/NAS. Nutrient requirements of the laboratory rat. In: National Research Council, National Academy of Sciences. Nutrient Requirements of Laboratory Animals, 3 rd edition, Washington, D. C. 1978:7-37.

22. Block R J, Weiss KW. Methods and results of protein analysis. In: Block R J, Weiss KW, ed. Amino Acid Handbook Spingfield, Illinois: Charles C. Thomas, 1956 pp. 272.

23. Waterlow JC, Garlick PJ, Millward DJ. The precursor pool for protein synthesis. in: Waterlow JC, Garlick P J, Millward D J, ed. Protein" Turnover in Mammalian Tissues and in the Whole Body, Amsterdam: Elsevier/North-Holland Biomedical Press, 1978:145-164.

24. Mitlward DJ, Garlick P J, Nnanyeguzo DO, Waterlow JC. The relative importance of muscle protein synthesis and breakdown in the regulation of muscle mass. Biochem J 1976; 156:185-188.

25. Pain VM, Garlick PJ. Effect of streptozotocin diabetes and insulin treatment on the rate of protein synthesis in tissues of the rat in vivoo J Biol Chem 1974; 249:4510-4514.

26. Smith CK, Durschlag RP, Layman DK. Response of skeletal muscle protein synthesis and breakdown to levels of dietary protein and fat during growth in the weanling rat. J Nutr 1982; 112:255-262.

27. Li JB, Goldberg AL. Effects of food deprivation on protein synthesis and degradation in rat skeletal muscles. Am J Physiol 1976; 231:441-448.

28. Fajans SS, Quibrera S, Pek S, Floyd JC, Christensen HN, Jr., Conn JW. Stimulation of insulin release in the dog by a non-metabolizable amino acid comparison with leucine and arginine. J Clin Endocrinol Metab 1971; 33:35-41

29. Schwenk WF, Haymond MW. Effects of leucine, isoleucine, or threonine infusion on leucine metabolism in humans. Am J Physiol 1987; 254:E428-E434.

30. Frexes-Steed M, Warner ML, Bulus N, Flakoll P, Abumrad NN. Role of insulin and branched-chain amino acids in regulating protein metabolism during fasting. Am J Physiol 1990; 258:Eg07-E917.

31. Tessari P, Inchiostro S, Biolo G, Trevisan R, Fantin G, Marescotti MC, lore E, Tiengo A, Crepaldi G. Differential effects of hyperinsulinemia and hyperaminoacidemia on leucine-carbon metabolism in vivo. J Clin Invest 1987; 79:1062-1069.

32. Castellino P, Luzi L, Simonson DC, Haymind M, DeFronzo RA. Effect of insulin and ptasma amino acid concentrations on leucine metabolism in man. J Clin Invest 1987; 80:1784-1793.

Accepted for publication July 13, 1991.