Department of Exercise Science and Physical Education, University of Iowa, Iowa City, Iowa ... Thirty minutes after treadmill exercise of high and moderate.
Effect of insulin on protein synthesis in skeletal muscle after exercise THOMAS MICHAEL
and degradation
W. BALON, ANTONIO ZORZANO, JUDITH N. GOODMAN, AND NEIL B. RUDERMAN
L. TREADWAY,
Department of Exercise Science and Physical Education, University of Iowa, Iowa City, Iowa 52242; Division of Diabetes and Metabolism, Evans Memorial Department of Medicine, University Hospital, and Department of Physiology, Boston University Medical Center, Boston, Massachusetts 02118; Departamento de Bioquimica y Fisiologia, Facultad de Biologia, Universidad de Barcelona, Barcelona 08071, Spain; and Department of Medicine, Division of Endocrinology, University of California at Davis, Sacramento, California 95817 BALON, THOMAS W., ANTONIO ZORZANO, JUDITH L. TREADWAY, MICHAEL N. GOODMAN, AND NEIL B. RUDERMAN. Effect of insulin on protein synthesis and degradation in skeletal muscle after exercise. Am. J. Physiol. 258 (Endocrinol. Metab.
21): E92-E97, 1990.-This study examined whether insulin stimulation of protein synthesisand inhibition of protein degradation is enhancedafter exercise. The isolated perfused rat hindquarter preparation wasusedto evaluate net protein breakdown, myofibrillar protein degradation, and protein synthesis. Thirty minutes after treadmill exercise of high and moderate intensity, rates of tyrosine releasewere increased by 58 and 25%, respectively. Insulin at 75 pU/ml had no effect on these increasesafter intense exercise; however, 20,000 pU/ml of insulin totally inhibited this increase.Cycloheximide increased the tyrosine releasein both control and exercised rat muscle. It also abolishedthe difference between them, suggestingthat the increasein tyrosine releaseafter exercise is causedby an inhibition of protein synthesis. Phenylalanine incorporation into protein was marginally depressed(22%, P = NS) in the white gastrocnemiusmuscleafter intense exercise. Insulin at 200 pU/ml stimulated protein synthesis in these rats, but no more than it did in a nonexercised control group. Failure to observea greater effect of insulin on protein metabolism was also noted when rat musclewas studied 150 min after intense exercise and after contractions induced by electrical stimulation of the sciatic nerve. These findings suggestthat after exerciseor electrically induced contractions the enhancedability of insulin to stimulate hexose and amino acid transport is not paralleled by an increasein its ability to stimulate protein synthesisor inhibit protein degradation. cycloheximide; hindquarter; perfusion; phenylalanine; rat; tyrosine; 3-methylhistidine
REGULATES a myriad of metabolic processes in skeletal muscle including glucose and amino acid transport, potassium uptake, and glycogen synthesis and degradation. Insulin also appears to increase the synthesis of both sarcoplasmic and ribosomal proteins by facilitating the translation of mRNA (22). While the exact mechanism(s) of insulin’s stimulatory effect on protein synthesis is still unknown, insulin could modulate the availability of amino acids, high-energy phosphates, ribosomes, tRNA, and mRNA, and/or regulate the activity INSULIN
E92
of enzymes and factors that catalyze peptide formation, peptide-chain initiation, elongation, and termination (for review, see Ref. 19). In mammals, protein catabolism appears to be primarily under the control of decreased insulin and secondarily under permissive effects of glucocorticoids (8), which in turn are dependent on a number of factors including nutritional and activity status. The ability of insulin to stimulate glucose (27) and amino acid transport (35, 36) is increased after exercise. Whether protein metabolism is similarly affected is unclear. Protein synthesis and degradation have been reported to be increased, decreased, and unchanged after exercise. Possible reasons for this variability include differences in age, evaluation model, nutritional state, exercise type, duration, and intensity, and the time lapse after exercise when protein metabolism was evaluated (6, 7, 9, 11, 12, 18, 21). The effects of insulin on protein metabolism after exercise are also unclear. The present study addresses these questions by utilizing experimental systems and protocols with which we have previously demonstrated a well-defined and replicable enhancement of insulin on other metabolic processes after exercise. Protein synthesis, net protein catabolism, and myofibrillar protein degradation were measured after various exercise regimens using the perfused rat hindquarter preparation. The results indicate that net protein catabolism is increased after exercise and that the increase is transient and related to the intensity of prior exercise. Our findings also suggest that in contrast to hexose and amino acid transport, prior exercise does not enhance the ability of insulin to stimulate protein synthesis or inhibit protein degradation. METHODS
AnimaZs. Male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing 200-260 g and fed Purina Laboratory Chow ad libitum were used in all experiments. To accustom them to treadmill running, rats were run for 5 min at a rate of 36 m/min for 3 days during the week prior to the experiment. On the morning of the study the rats were randomly assigned to either a control (nonexercised) or exercise group. The exercised rats ran
0193~1849/90 $1.50 Copyright 0 1990 the American Physiological Society
MUSCLE
PROTEIN
METABOLISM
on a motor-driven treadmill according to one of the following protocols: 18 m/min for 43 min followed by 2 min at 36 m/min (moderate-intensity exercise) or 36 m/ min for 50 min in lo-min bouts interspersed with 3 min of rest (high-intensity exercise). Perfusion. Immediately after the run the rats were anesthetized with pentobarbital sodium (5 mg/lOO g body wet wt ip) and surgically prepared for perfusion by the method of Ruderman et al. (29) as modified by Goodman et al. (17). The initial perfusion medium consisted of Krebs-Henseleit solution, aged-rejuvenated human erythrocytes (30% hematocrit), 4% bovine serum albumin (Cohn fraction V, Pentex, Kankakee, IL), 6 mM glucose, and 0.15 mM pyruvate. In all studies of protein synthesis and in other cases as indicated, physiological amounts of amino acids (23) and 0.8 mM phenylalanine were added to the perfusate. The initial volume of perfusate was 150 ml. After surgical preparation, the hindquarter was immediately placed in a perfusion cabinet maintained at 37°C.. The first 25 ml of perfusate passing through the preparation were discarded and the perfusate was then recycled. At this time, insulin (crystalline pork insulin, 617-07-256, courtesy Eli Lilly, Indianapolis, IN) was added to the perfusate. The hindquarter was then perfused for 15 min to allow for equilibration prior to experimental measurements and/or other manipulations (e.g., electrical stimulation). Electrical stimulation. The procedure for inducing muscle contractions by electrical stimulation of both sciatic nerves has been described previously (4, 28). In the present study, immediately following the 15 min of equilintracellular
sp act (dpm/pmol)
Phe incorporation
into protein
AFTER
EXERCISE
methylhistidine determination, perfusate samples (2 ml) were deproteinized with 70 ~1 of 60% PCA. Evaluation of protein synthesis. Protein synthesis was assessed using a modification of the method described by Jefferson and co-workers (20) for the perfused rat hemicorpus preparation. Ten microcuries of L-[4-3H]phenylalanine and 0.8 mM phenylalanine were added to the initial medium after a 15-min equilibration period. The extracellular space (ECS) of the white gastrocnemius and soleus were assumed to be 10 and 17%, respectively (36) In all studies of protein synthesis, the soleus and the white portion of the gastrocnemius were removed from the left leg and frozen in liquid nitrogen 30 min after the addition of the phenylalanine. The remainder of the lower left leg was tied off to minimize blood loss. The same muscles were removed from the right leg after an additional 30 min of perfusion. The muscles were then weighed and homogenized in 3 ml of 6% ice-cold perchloric acid with a polytron homogenizer. The homogenate was centrifuged and the supernatant collected. The protein pellet was washed with an additional 2 ml of 6% ice-cold perchloric acid and recentrifuged. The supernatants were then combined and assayed for phenylalanine. The pellet was dried, transferred to a liquid scintillation vial, and dissolved in NCS (Amersham, Arlington Heights, IL). Ten milliliters of Scintiverse II (Fisher, Fairlawn, NJ) were added to the vial and counted in a Beckman model LS 7000 liquid scintillation counter. The incorporation of [3H]phenylalanine into protein and of the intracellular specific activity (sp act) of phenylalanine (Phe) were calculated as follows
=
[14C]Phe (dpm/g tissue) - ( [ 14C]Phe (dpm/ml perfusate) x ECS (ml/g)] Phe (pmol/g tissue) - [Phe (pmol/ml perfusate) X ECS (ml/g)]
=
(dpm/g muscle at 60 min) - dpm/g muscle at 30 min (intracellular sp act at 30 min + intracellular sp act at 60 min)/2
ibration, the preparation was stimulated for two 5-min periods separated by 1 min of rest. Each stimulation period consisted of one 500-ms train/s with each train consisting of repeated 6- to 8-V pulses of O.l-ms duration delivered at 100 Hz. Perfusion flow rate was increased from 12.5 to 25 ml/min during the 11-min stimulation period to assure adequate oxygenation. Glucose uptake and other measurements were performed during the 30 min after the cessation of stimulation. Evaluation of protein degradation. Net protein breakdown and total protein breakdown were estimated by measu ring the release of tyrosine during 30 min of hindquarter perfusion in either the absence (net) or presence (total) of 200 PM cycloheximide. Myofibrillar protein breakdown was assessed by 3-methylhistidine release from the hindquarter during 30 min of perfusion in the absence or presence of 200 PM cycloheximide. At this concentration of cycloheximide, protein synthesis is inhibited by at least 90%. Perfusate samples (1 ml) were collected from the reservoir, deproteinized with 2 ml of 6% perchloric acid (PCA), centrifuged at 4°C for 30 min (2,500 g), and frozen until analyzed for tyrosine. For 3-
E93
where dpm is disintegrations per minute. Analytical procedures. Tyrosine was assayed fluorometrically by the method of Waalkes and Udenfriend (31) and 3-methylhistidine by a slight modification of the high-performance liquid chromatography method (25) as originally described by Wassner and co-workers (33). Phenyla lanine was measured fluorometrically by the procedure of McCama .n and Robins (26) as modified by Andrews et al. (2). At the same time that muscle samples were obtained, samples of the perfusion medium (whole blood) were deproteinized in 6% cold perchloric acid and centrifuged (4°C). The supernatant was anaa.nd radioactivity. 1Yvzed for phenylalanine 1concentration Statistics. Statistical analysis was by an alysis of variante with a subsequent Scheffe or Newman-Keuls post hoc comparison. RESULTS
Tyrosine release after exercise. Tyrosine is neither catabolized nor synthesized by mu scle; therefore, its release into the perfusate provides l a measure of the balance between protein synthesis and degradation (net protein
E94
MUSCLE
PROTEIN
METABOLISM
breakdown) by the hindquarter preparation (13, 16). As shown in Table 1, the release of tyrosine was increased 30 min after exercise of moderate intensity, and to an even greater extent after high-intensity exercise. By 150 min after the cessation of high-intensity exercise, tyrosine release had returned to control values, indicating that this postexercise increase is transient. Insulin, at concentrations shown previously to stimulate glucose and a-aminoisobutyrate transport after exercise (75 pU/ml) (27, 35), did not significantly decrease tyrosine release in any of the groups. Significant decreases were seen, however, when the hindquarter was perfused with 20,000 pU/ml of insulin. At this concentration insulin decreased tyrosine release most markedly in the hindquarters of the previously exercised rats; indeed, the increase in tyrosine release observed after both moderate- and high-intensity exercise was totally eliminated. Studies at intermediate concentrations of insulin were not carried out. The possibility that the increase in tyrosine release after exercise is caused by a “washout” of this amino acid from muscle was next examined. As shown in Table 2, intramuscular tyrosine levels were increased immediately after the cessation of intense exercise; however, by 60 min they were no different from those of nonexercised rats. More importantly, as shown in Table 3, the concentration of tyrosine, although increased modestly in some hindlimb muscles at the time of perfusion, did not di-
1. Effects of prior exercise and of insulin on tyrosine releasefrom perfused rat hindquarter TABLE
AFTER
EXERCISE
TABLE 3. Effects of perfusion on concentration of tyrosine in selected musclesof perfused hindquarter Tyrosine, Soleus
Min of perfusion Nonexercised PE-30
0 68t6 62t3
nmol/g White gastrocnemius
30 68t3 72t4
0 68k8 86&3*
30 64k2 79,t6*
Values are means k SE of 5-8 experiments. Hindquarters of sedentary or previously exercised rats run on treadmill at high intensity were allowed to equilibrate for 15 min in the perfusion chamber before initial sampling of tissue. See METHODS and Table 1 for details. * Significantly different from nonexercised control, P < 0.05.
4. Effect of electrically induced contractions on tyrosine release by perfused rat hindquarter
TABLE
Tyrosine
Release, nmol 6g-l. h”
Control
Poststimulation
Insulin 0 pU/ml 51&4 5Ok7 45&11 45k4 75 pU/ml Values are means & SE of 5-8 observations. After 15 min of equilibration, both hindlimbs were made to contract by electrical stimulation of the sciatic nerve (500-ms trains) at a rate of l/s for a total of 10 min (see METHODS). Tyrosine release was measured during the 30 min after stimulation.
5. Effects of prior intense exercise and cycloheximide on release of tyrosine and 3-methylhistidine by perfused rat hindquarter
TABLE
Release, nmol g” l
Exercise Intensity Moderate Control
3-Methylhistidine Intense
PE-30 Tyrosine
Control release,
PE-30 nmol
lg-l
PEG0 l
h-l
PE-30
Insulin 0 pU/ml 54t4 67zk5* 55t5 87k5” 51t4 75 pU/ml 51t7 6Ozk4 56&3 7227” 59zk5 20,000 pU/ml 46t9 44+5-f 40+3t 47a7t 39&2? Values are means k SE of 5-8 observations. Rats were run on treadmill at moderate or high intensities (see METHODS). At either 30 or 150 min postexercise (PE), hindquarters were perfused with medium to which amino acids were not added. Tyrosine release was measured over 30 min after a 15-min equilibration period, and results were corrected for mass (g-l, wet wt) of skeletal muscle perfused as described (29). * Values significantly different from nonexercised controls. t Values significantly different from comparable groups perfused in absence of insulin (P < 0.05).
2. Tyrosine concentration in blood and selected muscles after high-intensity exercise
TABLE
Control
Min After Exercise 0
Addition to perfusate None Control
60
Blood 54t2 107*20* 54t5 Soleus 91t7 128+15t 103k13 Red gastrocnemius 99&S 120+7p 89k7 White gastrocnemius 105*7 146+23t 99k7 Values are means & SE with 5-8 observations/group. Blood and tissue concentrations are in nmol/ml and nmol/g, respectively. Samples were taken from anesthetized rats at indicated times after treadmill run and from control rats that had not exercised. Significantly different from control: * P < 0.01; t 0.05.
1.33*0.10 1.62kO.09”
l
h-’ Tyrosine
55+5 87*5*
Cycloheximide, 0.2 mM 1.33kO.07 118&8 Control 1.68kO.21 123t2 PE-30 Values are means k SE of 5-8 observations. At concentration used, cycloheximide inhibited phenylalanine incorporation into protein by 90%. See Table 1 and METHODS for details. * Significantly different (P < 0.05) from control.
minish over the 30 min during which its release into the perfusate was measured. Tyrosine release after electrically induced contractions.
It has previously been shown that after intense treadmill exercise or 10 min of electrically induced contractions, hexose and AIB transport by the perfused hindquarter are increased, as is the ability of insulin to stimulate these processes (15,27,28,37). The high-intensity treadmill run and electrical contractions used in this study both result in a 60-70% depletion of muscle glycogen levels (15, 28). However, electrical stimulation did not increase the release of tyrosine by the hindquarter in either the presence or absence of insulin (Table 4), in contrast to the results obtained after a high-intensity treadmill run (Table 1). Degradation of myofibrillar and nonmyofibrillar protein. As suggested by earlier studies, the release of 3-
methylhistidine
by the hindquarter
provides an index of
MUSCLE PROTEIN METABOLISM TABLE 6. Effects of prior intense exercise and of insulin on protein synthesis Soleus Control
White Gastrocnemius
Exercised
Phenylalanine
Insulin PE-30 0 j&J/ml 200 pU/ml
PE-150 0 &J/ml
Control
incorporation nmol g-l . h-’
into protein,
l
118k12 156t13*
Exercised
77k9
121t16 170t14*
6Ok3
108t8*
90t11*
89k5 88k7 58k9 66t6 200 pU/ml 106&7* 112k7* 79*5* 78t8 Values are means k SE of 5-9 observations. Perfusate contained physiological concentrations of amino acids and 0.8 mM [4-3H]phenylalanine. Specific activity of phenylalanine in muscle was the same in all control and exercised groups. * Significantly different (P < 0.05) vs. group perfused in absence of insulin. See METHODS for details.
TABLE 7. Effect of perfusion with physiological concentrations of amino acids and 0.8 mM Phe on muscle Tyr concentration and release of Tyr by isolated perfused rat hindquarter
AFTER EXERCISE
E95
ylalanine incorporation into protein was unchanged 30 min after intense exercise in the soleus. It was slightly diminished by 22% in the white fibers of the gastrocnemius, although the decrease was not statistically significant (0.05 c P < 0.11). No differences were observed 150 min after exercise in either muscle. Insulin at a concentration of 200 &J/ml stimulated phenylalanine incorporation into protein to a comparable degree in all groups, suggesting that its ability to alter protein synthesis was not altered by prior exercise. As in the hindquarters perfused without insulin, the rate of phenylalanine incorporation was slightly (17%), but not significantly, lower in the white gastrocnemius in the exercised group. Data for the release and intramuscular concentrations of tyrosine in these studies are presented in Table 7. Absolute rates of tyrosine release were diminished (compare with Table l), presumably because of the addition of amino acids to the perfusate (23); however, the difference between the nonexercised and exercised groups persisted. The intramuscular concentrations of tyrosine were higher in the exercised groups in these studies; however, there was no loss of tyrosine from either the soleus or gastrocnemius during the perfusion (Table 7).
AA + 0.8 mM Phe Min of Perfusion
Soleus 0 Muscle
Nonexercised After intense exercise, 30 min
96t4 146t14*
White gastrocnemius 30
tyrosine
0 concentration,
117t3 166+25*
Tyrosine
PE-30 release,
nmol jg
90t5 135*15*
AA + 0.8 mM Phe Nonexercised
30
10529 128t7*
No Additions Nonexercised nmol
lg-l
PE-150 eh-’
Insulin 0 pU/ml 25t6 58-1-5t 53k4 83+5t 200 pU/ml 15k5 39*9* Values are means t SE of 5-8 observations. See legend to Tables 1 and 6 and METHODS for details. Values significantly different from nonexercised control rats: * P < 0.05 and t P < 0.0.1. AA, amino acids, Phe, [4-3H]phenylalanine.
myofibrillar protein degradation (24, 32). Tyrosine release, however, reflects the average rate of degradation of both myofibrillar and nonmyofibrillar protein pools. The data in Table 5 demonstrate that the release of both 3-methylhistidine and tyrosine is increased after intense exercise. The increase in 3-methylhistidine release is small, however, and the possibility that it could have been caused in part by a differential washout of 3methylhistidine from muscle was not examined. The addition of cycloheximide had no effect on the release of 3-methylhistidine by the hindquarter. Cycloheximide caused a marked increase in the release of tyrosine in all groups, and the differences between the control and exercised rats were no longer seen. Protein synthesis. Protein synthesis was evaluated directly in separate experiments in which physiological concentrations of amino acids and 0.8 mM [4-3H]phenylalanine were added to the perfusate (Table 6). Phen-
DISCUSSION
The ability of insulin to stimulate glucose and AIB uptake is markedly enhanced in skeletal muscle after exercise (3, 27, 35). In the present study, conditions identical to those used in earlier investigations (i.e., 30 min after moderate-intensity exercise and 30 and 150 min after high-intensity exercise) were used to examine the effects of prior exercise on protein synthesis and degradation. The results indicate that at the insulin concentration used (200 pU/ml), the ability of prior exercise to stimulate protein synthesis is not enhanced. Thus the stimulation of phenylalanine incorporation into protein by insulin was almost identical in nonexercised rats and rats studied 30 and 150 min after high-intensity exercise. Insulin significantly diminished the increase in net protein breakdown (tyrosine release) observed 30 min after exercise but only when present at a supraphysiological concentration (Table 1). Furthermore, at 150 min after high-intensity exercise, when increases in the sensitivity of the glucose and AIB transport systems to insulin are still demonstrable, insulin suppression of tyrosine release by control and previously exercised skeletal muscle did not differ. These findings collectively suggest that prior exercise does not alter a very early step in insulin action, but that it selectively alters the signals generated by insulin in the muscle cell. The site and mechanisms by which prior exercise selectively enhances insulin signaling in skeletal muscle are unknown. The site appears to be distal to the insulin receptor because we have found that prior exercise alters neither insulin binding (35) nor activation of the intrinsic protein tyrosine kinase of the P-subunit of the insulin receptor (30). It is unlikely that the effects of insulin observed in this‘study were mediated through the insulinlike growth factor I receptor because of the extremely low affinity of insulin binding to the insulin-like growth
E96
MUSCLE
PROTEIN
METABOLISM
factor receptor (1,5). It has also been demonstrated that prior exercise inhibits insulin stimulation of prostaglandin release from the hindquarter preparation (38); however, the mechanistic significance of this finding remains to be determined. The effects of prior exercise on the sensitivity and responsiveness of protein synthesis and net protein degradation in muscle to insulin have also been examined by Davis and Karl (10) using an incubated epitrochlearis preparation. They found that net protein breakdown increases and that protein synthesis decreases after acute exercise (2 h of swimming). In contrast to the findings of the present study, the authors reported that the sensitivity of protein synthesis to insulin is increased after exercise. The relevance of these findings to those presented here is unclear, since the experimental conditions were different. Furthermore, the increase in sensitivity to insulin was evaluated in preparations that displayed impaired protein synthesis after exercise, and it was not observed in all experimental groups. Our findings indicate that net protein degradation is only transiently increased by prior exercise in the intact perfused rat hindquarter preparation. In keeping with earlier observations (11, 12), they also suggest that the magnitude of the increase correlates with the intensity of the exercise and that it is caused in part by a diminished rate of protein synthesis (11, 12). Other reports indicate that the rates of protein synthesis and degradation are decreased during electrically induced contraction in the rat hindquarter preparation (7). Interestingly, electrical stimulation of the rat hindquarter musculature in situ results in the same increase in insulin sensitivity of the glucose and AIB transport systems as in vivo treadmill exercise (28,37), but it does not result in a concomitant increase in net protein breakdown. This suggests that the enhanced breakdown of muscle protein after exercise is modulated by systemic and/or hormonal factors rather than by a factor generated by local muscle contractions. The hormonal mileu observed during exercise is consistent with this notion, as insulin concentration is markedly decreased, whereas glucocorticoid concentration is increased (l4), potentially favoring a net catabolic state. To examine the basis for the increase in net protein degradation in skeletal muscle following exercise, perfusions were performed in the presence of cycloheximide, an agent that inhibits protein synthesis at the level of ribosomal translation of mRNA. Cycloheximide increased the rate of release of tyrosine, but not 3-methylhistidine, in both control and previously exercised muscle. The difference in net protein breakdown (tyrosine release) between the exercised and control rats was abolished, suggesting that the transient increase in net protein breakdown after exercise predominantly results from a decrease in protein synthesis. This conclusion should be interpreted with caution, however, since cycloheximide may independently diminish protein degradation to a small extent (34). In summary, the increased ability of insulin to stimulate glucose and amino acid transport after exercise is not naralleled bv effects on nrotein metabolism. Earlier
AFTER
EXERCISE
studies have shown that neither insulin binding nor insulin receptor tyrosine kinase activity are altered after the same exercise regimen. Based on these findings, we conclude that prior exercise selectively enhances specific signals generated by insulin in skeletal muscle. The authors thank Joan A. Seye for secretarial assistance. This work was supported in part by National Institutes of Health Grants AM-19154 to N. B. Ruderman, AM-19469 to M. N. Goodman, and T-AM-00652, and University of Iowa Old Gold Summer Fellowship and American Diabetes Association Feasibility Grant to T. W. Balon. Address for reprint requests: T. W. Balon, Dept. of Exercise Science and Physical Education, Univ. of Iowa, Iowa City, IA 52242. Received 5 June 1989; accepted in final form 29 August 1989. REFERENCES 1. ALEXANDRIDES, T., A. C. MOSES, AND R. J. SMITH. Developmental expression of receptors for insulin, insulin-like growth factor I (IGF-I) and IGF-II in rat skeletal muscle. Endocrinology 124: 10641076,1989. 2. ANDREWS, T. M., R. GOLDTHORP, AND R. W. E. WATTS. Fluorimetric measurement of the phenylalanine content of human granulocytes. Clin. Chem. Actu 43: 379-387, 1973. 3. BALON, T. W., A. ZORZANO, M. N. GOODMAN, AND N. B. RUDERMAN. Insulin increases thermogenesis in rat skeletal muscle following exercise. Am. J. Physiol. 248 (Endocrinol. Metub. 11): E148-E151,1985. 4. BALON, T. W., A. ZORZANO, M. N. GOODMAN, AND N. B. RUDERMAN. Insulin-enhanced thermogenesis in skeletal muscle following exercise: regulatory factors. Am. J. Physiol. 251 (Endocrinol. Metub. 14): E294-E298, 1986. 5. BEGUINOT, F., C. R. KAHN, A. C. MOSES, AND R. J. SMITH. Distinct biological active receptors for insulin, insulin-like growth factor I, and insulin-like growth factor II in cultured skeletal muscle cells. J. Biol. Chem. 260: 15892-15898, 1985. 6. BOOTH, F. W., W. F. NICHOLSON, AND P. A, WATSON. Influence of muscle use on protein synthesis and degradation. In: Exercise and Sport Sciences Reviews, edited by R. L. Terjung. Philadelphia, PA: Franklin Institute, 1982, vol. X, p. 27-48. 7. BYLUND-FELLENIUS, A. C., K. M. OJAMAA, K. E. FLAIM, J. B. LI, S. J. WASSNER, AND L. S. JEFFERSON. Protein synthesis versus energy state in contracting muscles of perfused rat hindlimb. Am. J. Physiol. 246 (Endocrinol. Metub. 9): E297-E305, 1984. 8, CAHILL, G. F., JR., T. T. AOKI, AND E. B. MARLISS. Insulin and muscle protein. In: Handbook of Physiology. Endocrinology. Endocrine Pancreas. Washington, DC: Am. Physiol. SOC., 1972, sect. 7, vol. I, chapt. 37, p. 563-577. 9. CLARK, A. S., AND W. E. MITCH. Comparison of protein synthesis and degradation in incubated and perfused muscle. Biochem. J. 212: 649-653,1983. 10. DAVIS, T. A., AND I. E. KARL. Response of muscle protein turnover to insulin after acute exercise and training. Biochem. J. 240: 651657,1986. 11. DOHM, G. L., G. J. KASPEREK, E. B. TAPSCOTT, AND G. R. BEEDNER. Effect of exercise on synthesis and degradation of muscle protein. Biochem. J. 188: 255-262, 1980. 12. DOHM, G. L., E. G. TAPSCOTT, AND G. J. KASPEREK. Protein degradation during endurance exercise and recovery. Med. Sci. Sports Exert. 19, Suppl: S166-S171,1987. 13. FULKS, R. M., J. B. LI, AND A. L. GOLDBERG. Effects of insulin, glucose and amino acids on protein turnover in rat diaphragm. J. Biol.
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M. N., N. B. RUDERMAN, AND T. AOKI. Glucose and amino acid metabolism in perfused skeletal muscle. Effect of dichloroacetate, Diabetes 1065-1074, 1978. GOODMAN, M. N. Amino acid and protein metabolism. In: Exercise, Nutrition und Energy Metabolism, edited by E. S. Horton and R. L. Terjung. New York: Macmillan, 1988, p. 89-99. JEFFERSON, L. S. Role of insulin in the regulation of protein synthesis. Diabetes 29: 487-496, 1980. JEFFERSON, L. S., J. B. LI, AND S. R. RANNELS. Regulation by insulin of amino acid release and protein turnover in the perfused rat hemicorpus. J. Biol. Chem. 252: 1476-1483,1977. KASPEREK, G. J., AND R. D. SNIDER. The effect of exercise on protein turnover in isolated soleus and extensor digitorum longus muscles. Experientia Busel41: 1399-1400, 1985. KURIHARA, K., AND I. G. WOOL. Effect of insulin on the synthesis of sarcoplasmic and ribosomal proteins of muscle. Nature Lond.
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AND M. N. GOODMAN. Evidence that lysosomes are not involved in the degradation of myofibrillar proteins in rat skeletal muscle. Biochem. J. 234: 237-240, 1986. 25. LOWELL, B. B., N. B. RUDERMAN, AND M. N. GOODMAN. Regulation of myofibrillar protein degradation in rat skeletal muscle during brief and prolonged starvation. Metabolism 35: 1121-1127, 1986. 26. MCCAMAN, M, W., AND E. ROBINS. Fluorimetric method for the determination of phenylalanine in serum. J. Lab. Clin. Med. 59: 885-890,1962. 27. RICHTER, E. A., L. P. GARETTO, M. N. GOODMAN, AND N. B. RUDERMAN. Muscle glucose metabolism following exercise in the rat: increased sensitivity to insulin. J. Clin. Inuest. 69: 785-793, 1982. 28. RICHTER, RUDERMAN.
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29. RUDERMAN, N. B., C. R. S. HOUGHTON, AND R. HEMS. Evaluation of the isolated perfused hindquarter in the study of muscle metabolism. Biochem. J. 124: 639-651,197l. 30. TREADWAY, J. L., D. E. JAMES, E. BURCEL, AND N. B. RUDERMAN. Effect of exercise on insulin receptor binding and kinase activity in skeletal muscle. Am. J. Physiol. 256 (Endocrinol. Metub. 19): El38-El44,1989. 31. WAALKES, T.
P., AND S. UDENFRIEND. A fluorometric method for the estimation of tyrosine in plasma and tissues. J. Lab. Clin. Med.
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