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Effect of catabolic hormone infusion on protein turnover and amino acid uptake in skeletal muscle
Effect of Catabolic Hormone Infusion on Protein Turnover and Amino Acid Uptake in Skeletal Muscle Brad W. Warner, MD, Per-Olof Hasselgren, MD, Robert P. Hummel III, MD, J. Howard James, BS, Peter Pedersen, MD, Josef E. Fischer, MD, eAES, Cincinnati,Ohio
Increased plasma levels of the catabolic hormones glucagon, epinephrine, and cortisol have been implicated in mediating various metabolic alterations in trauma and sepsis. Their role in altered protein turnover and amino acid transport in skeletal muscle during sepsis, however, is not known. In the current study, rats were infused with a mixture of the catabolic hormones for 16 hours. Control animals were infused with vehicle solution. Protein synthesis and degradation rates were measured in incubated, intact soleus muscles as incorporation of 14C-phenylalanine into protein and release of tyrosine into incubation medium, respectively. Muscle amino acid uptake was determined by measuring the intracellular to extracellular ratio of [3H]-c~-aminoisobutyric acid after incubation for 2 hours. Infusion of catabolic hormones for 16 hours resulted in elevated plasma glucose and lactate levels, reduced plasma concentrations of most amino acids, and accelerated muscle protein breakdown, similar to previous findings in septic rats. Protein synthesis rates and amino acid uptake in incubated muscles were not significantly different in control and hormoneinfused rats. The current study suggests that increased muscle proteolysis in sepsis and severe injury may be mediated in part by catabolic hormones. In contrast, reduced muscle protein synthesis and amino acid uptake are probably signaled by other substances or mechanisms.
From the Department of Surgery, Universityof Cincinnati Medical Center, Cincinnati, Ohio. Supportedin part by grant 1R01 DK3790801 fromthe National Institutes of Health, Bethesda,Maryland. Requests for reprintsshould be addressedto JosefE. Fischer,MD, Department of Surgery,Universityof Cincinnati Medical Center, 231 BethesdaAvenue(ML# 558), Cincinnati,Ohio45267. Manuscript submittedAugust 23, 1988,revisedMarch 18, 1989, and acceptedApril 5, 1989.
evere trauma and sepsis are characterized by accelerated degradation and reduced synthesis of skeletal muscle protein [1-4] and by inhibition of muscle amino acid uptake [5,6]. The mediators of these metabolic responses to sepsis are currently unknown. Plasma levels of the "catabolic hormones" glucagon, epinephrine, and cortisol are elevated after severe injury or sepsis [7-9], and administration of these hormones to normal subjects results in metabolic alterations commonly observed in stressed patients, such as hypermetabolism, negative nitrogen balance, hyperglycemia, and peripheral insulin resistance [10-12]. Consequently, the catabolic hormones have been implicated in mediating various metabolic alterations in trauma and sepsis, but their role in altered muscle protein turnover and amino acid transport is not known. The purpose of the current study was to determine the effect of catabolic hormone infusion in rats on protein synthesis and degradation and amino acid uptake in skeletal muscle.
S
MATERIAL AND M E T H O D S Male Sprague-Dawley rats weighing approximately 60 g were used. Animals of this size possess thin soleus muscles suitable for diffusion of nutrients and oxygen during in vitro incubation [13]. After acclimatization for 2 to 3 days in a room with a 12-hour light/12-hour dark cycle (240C), rats underwent central venous catheterization. With rats under pentobarbital anesthesia (45 mg/kg intraperitoneally), the neck and back regions were shaved and scrubbed with a povidone-iodine solution. An external jugular vein was cannulated with Silastic tubing (0.025-inch inner diameter), which was passed subcutaneously to exit the mid-scapular region of the back. The catheter was protected externally by a coiled steel spring attached to a swivel apparatus. After catheterization, animals were housed in individual metabolic cages and were infused with catabolic hormones or vehicle solution for 16 hours. Water, but no food, was provided during the period of hormone or control infusion. At the end of the infusion, soleus muscles were removed for studies of protein turnover and amino acid uptake. Blood was obtained by heart puncture for determination of levels of plasma amino acids, glucose, lactate, and blood urea nitrogen. Care and handling of animals in all experiments conformed to the guidelines established by the Department of Laboratory Animal Medicine of the University of Cincinnati Medical Center. The current experimental protocol involves anesthesia, surgical incision of the neck, and 16-hour fasting. Although control and hormone-infused rats underwent
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identical treatment, it is possible that the different aforemtioned factors could alter basal protein turnover rates or amino acid uptake, which in turn could alter the response to hormone infusion. Control experiments were therefore performed in which muscle protein synthesis and degradation rates and amino acid uptake were measured in fed and 16-hour fasted rats and in rats that had been anesthetized (pentobarbita145 mg/kg) or subjected to skin incision of the neck 16 hours earlier. Rats were infused with a mixture of glucagon (2.5 gg/ kg/hour), epinephrine (6 tzg/kg/hour; adrenalin chloride), and corticosterone (4.2 mg/kg/hour) dissolved in normal saline and with the addition of ascorbic acid (1 mg/mL) and albumin (3 mg/mL). Control animals received an infusion of the same solution, but without hormones. Infusions were administered at a rate of 1.1 m L / hour for 16 hours. With the exception of glucagon, the amounts of hormone administered were the same as in a previous study from our laboratory in which the infusion resulted in plasma levels consistent with those reported in injured and septic patients [14]. In that study, plasma glucagon levels were higher than previously reported after injury and sepsis. In this study, therefore, the dose of glucagon administered was half of that administered in our previous report. In separate experiments, plasma levels of glucagon were determined [14] at the end of the infusion period. Animals in these experiments were somewhat larger (body weight approximately 120 g) than in the experiments in which muscles were incubated in vitro. Since the concentrations of corticosterone and epinephrine in the current infusion solution were identical to those in our previous report [ 14], measurements of plasma concentrations of these hormones were not repeated here. Amino acid uptake was determined in incubated soleus muscles by measuring the uptake of [JH]-a-aminoisobutyric acid (A1B), a synthetic amino acid analogue that is neither metabolized nor incorporated into protein [15] and is predominantly transported by system A [16,17]. With rats under light ether anesthesia, soleus muscles were removed with intact tendons and placed individually in 25-mL Erlenmeyer flasks containing 3 mL of oxygenated (O2:CO2 = 95:5) Krebs-Henseleit bicarbonate buffer (pH 7.4) with glucose (10 mmol/L), [JH]AIB (0.5/~Ci/mL, 0.5 mmol/L), and [~4C]-inulin (0.05 ~Ci/mL). The medium was gassed with O2:CO2 (95:5), the flask was stoppered, and the incubation was performed at 37~ for 2 hours in a shaking water bath. After incubation, muscles were blotted, weighed, and dissolved in 1 mL of tissue solubilizer and 0.1 mL of water. Radioactivity of dissolved muscles and media samples was measured in a scintillation spectrophotometer. Intracellular [3H]-AIB was determined from total tissue [JH]AIB corrected for [JH]-AIB in the extracellular volume (measured as [14C].inulin space) as described previously [18]. Total tissue water of incubated soleus muscles was determined in separate experiments from wet and dry weights and was 79%. The AIB uptake was expressed as the intracellular to extracellular ratio of [JH]-AIB. For the study of protein synthesis, the muscle tendons ")96
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were tied to stainless steel frames to approximate resting length and preincubated for 30 minutes in 3 mL of oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4) with glucose (10 mm01/L). The medium was gassed with O2:CO2 (95:5), the flask was stoppered, and the incubation was performed at 37~ in a shaking water bath. After preincubation, muscles were gently blotted and transferred to 3 mL of fresh medium of the same composition as described earlier, but which also contained [14C]-phenylalanine (0.05/zCi/mL; 0.5 mmol/L). After incubation for 2 hours, the muscles were homogenized in 10% trichloroacetic acid (TCA). The precipitate was washed twice with 10% TCA and once with ether-ethanol (1:1). The pellet was dried overnight at room temperature and dissolved in 0.1 mL of water and 1 mL of tissue solubilizer, and the radioactivity was measured. Since a high concentration of phenylalanine was used in the incubation medium, the extraceUular specific radioactivity of [~4C]-phenylalanine was used to calculate amino acid incorporation into protein [19,20]. For the study of protein degradation, the Krebs-Henseleit bicarbonate buffer was supplemented with glucose (10 mmol/L), albumin (2 g/L), and physiologic levels of amino acids (with the exception of tyrosine). Soleus muscles were stretched to resting length as described earlier, and after 30 minutes, preincubation muscles were transferred to fresh medium containing cycloheximide (0.5 mmol/L) in addition to the aforementioned supplements. Proteolytic rate was assessed as release of tyrosine into the incubation medium over 2 hours as previously described [4,21]. Levels of plasma glucose and lactate were determined using a YSI Model 23A glucose and lactate analyzer, and blood urea nitrogen was measured calorimetrically. Plasma amino acids were analyzed using a 121-MB amino acid analyzer. Results are presented as mean values 4- SEM. Student's t test or analysis of variance (ANOVA), followed by Tukey's test, was used when statistical comparisons were made. RESULTS All animals tolerated the 16-hour infusion well, and there was no mortality in control or hormone-infused rats. Infusion of catabolic hormones for 16 hours resulted in significant elevations of plasma glucose and lactate levels, whereas no difference in blood urea nitrogen was noted (Table I). At the end of the infusion period, the plasma concentration of glucagon was 104 4- 3 pg/mL in control (n = 5) and 238 4- 20 pg/mL in hormone-infused rats (n --- 5; p <0.01). The plasma concentrations of all nonessential amino acids, with the exception of glutamic acid, were reduced in hormone-infused rats (Table II). Of the essential amino acids, threonine, methionine, isoleucine, lysine, histidine, and arginine were reduced. In a previous study, plasma concentrations of most amino acids were reduced in septic rats as well, although the concentrations of some amino acids were increased, most notably those of phenylalanine, tyrosine, and histidine [4].
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TABLE I Plasma Glucose and Lactate Levels and BUN In Rats Receiving Catabolic Hormone or Control Infusion for 16 Hours
Glucose (mg/dL) Lactate (mg/dL) BUN (mg/dL)
Control (n = 7)
Catabolic Hormones (n = 7)
101 4- 2 26 4- 2 14 4- 1
199 4- 4* 33 4- 2* 17 4- 2
9 p <0.01 versus odntrol. BUN = blood urea nitrogen.
AIB uptake, expressed as the intracellular to extracellular distribution ratio, was higher in soleus muscles of catabolic hormone-infused rats than in muscles of control animals, but the difference between the two groups of animals was not statistically significant (Table III). Protein synthesis rates were almost identical in incubated soleus muscles from control and hormone-infused rats, whereas protein degradation, measured as release of tyrosine, was increased by approximately 20% in the hormone-treated animals (Table liD, In control experiments, no significant differences in AIB uptake or protein synthesis or degradation rates were noted between fed rats and rats that had been subjected to a 16-hour fast, anesthesia only, or skin incision of the neck (Table IV). However, there was a significant difference in AIB uptake between 16-hour fasted rats and rats that had been subjected to anesthesia (p <0.05 by ANOVA) (Table IV). COMMENTS The current study showed that infusion of the catabolic hormones glucagon, epinephrine, and corticosterone in rats for 16 hours stimulated muscle proteolysis, measured as release of tyrosine from incubated soleus muscles. In previous reports from our laboratory, tyrosine release from incubated soleus muscles was increased 16 hours after induction of sepsis in rats [4,22], Thus, in this respect, the metabolic response to catabolic hormone infusion was similar to the metabolic response to sepsis, although the increase in tyrosine release was more pronounced in septic than in hormone-infused animals, In contrast, changes in muscle protein synthesis and amino acid uptake observed in septic rats [4-6,22] were not duplicated by hormone infusion. Thus, it is possible that some, but not all, metabolic alterations in sepsis are partially mediated by the catabolic hormones. The current finding of increased plasma levels of glucose and lactate further supports the concept that catabolic hormones play a role in metabolic alterations in sepsis [4,23]. In previous studies, triple hormone infusion in humans [10-12] and in rats [14] resulted in increased urinary nitrogen excretion. The results of the current study indicate that negative nitrogen balance reflected muscle protein breakdown, although altered protein turnover rates in other organs and tissues may have contributed.
TABLE II Plasma Amino Acid Levels ( / ~ m o l / L ) In Rats Receiving Catabolic Hormone or Control Infusion for 16 Hours Control (n = 7) Essential Threonine Valine Methionine Isoleucine Leucine Phenylalanine Tryptophan Lysine
Histidine Arglnine Nonessential Aspartlc acid Hydroxyproline Serine Asparagine Glutamic acid Glutamine Proline Glycine Alanine Tyrosine
Catabolic Hormones (n = 7)
264 250 59 132 199 78 72 377 81 127
4444444444-
7 12 1 4 4 3 7 14 1 9
124 229 31 110 179 74 71 232 67 69
4- 81. 4- 15 4- 1T 4- 6* 4- 10 4- 2 4- 7 4- 221 4- 3 T 4- 51.
25 39 289 94 56 685 173 574 324 54
4444444444-
1 3 10 4 16 28 11 42 13 2
18 14 131 62 53 428 65 213 167 37
4- 11. 4- 11 4- 41 4- 3 T 4- 4 4- 251 4- 51 4- 10 t 4- 121 4- 2 T
* p <0.05 versus control. t p <0.01 versus control.
TABLE I I I Protein Degradation, Prcteln Synthesis, and AIB Uptake In Incubated Soleus Muscles from Rats Receiving Catabolic Hormone or Control Infusion for 16 Hours
AIB uptake (intracellular/extracellular distribution ratio) Protein synthesis (nmol Phe/g 9 2h) Protein degradation (nmol Tyr/g 9 2h)
Control (n = 7)
Catabolic Hormones (n = 7)
3.86 4- 0.63
5.16 4- 0.56
170 4- 20
170 4- 10
370 4- 10
440 4- 20*
* p <0.02 versus control. Phe -- phenylalanine; Tyr = tyrosine.
It should be noted that the present experimental protocol included anesthesia, skin incision of the neck for placement of a venous catheter, and a 16-hour fasting period. Although both control and hormone-infused rats were treated identically, it may be argued that anesthesia, surgical incision, or fasting could alter basal AIB uptake and protein turnover rates, thereby influencing the response to hormone infusion. Control experiments, however, did not support that concept, since anesthesia alone, skin incision, or a 16-hour fast did not result in significant changes compared with fed, untreated rats. We do not currently have an explanation for the high AIB uptake in animals that had been subjected to anesthesia only, but a
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TABLE IV A I B U p t a k e and Protein Synthesis and D e g r a d a t i o n in I n c u b a t e d Soleus Muscles 16-Hour Fasted
Fed Rats AIB uptake (intracellular/extracellular Protein synthesis (nmol Phe/g ~ 2h)
distribution ratio)
Protein degradation (nrnol Tyr/g 9 2h)
Rats
Anesthesia
(n=6)
(n = 6 )
(n=6)
6.03 4- 0.65 292 4- 15 202 4- 29
4.79 4- 0.29 225 4- 18 250 4- 33
8.14 4- 0.42" 250 -4- 33 266 4- 18
Neck Incision (n=6)
6,72 4- 0.29 259 4- 5 270 4- 7
9 p<0.05 versus 16-hour fasted rats by ANOVA. Phe = phenylalanine; Tyr = tyrosine.
recent study suggests that general anesthetics can affect muscle cell membrane functions [24]. We recently reported another study in which catabolic hormones were infused in rats [14]. That study, however, differs from the current report in several important aspects. In this study, animals were infused for 16 hours since altered protein turnover rates and amino acid uptake were observed 16 hours after induction of sepsis in rats [4,22]. In contrast, in our previous report, animals were infused for 72 hours [14], similar to human studies reported by Bessey et al [II]. The animals used in this study were smaller (approximately 60 g) than those used in our previous report (approximately 100 to 150 g) and muscles were studied in vitro, whereas AIB uptake was measured in vivo in our earlier paper [14]. Small muscles are required for in vitro incubations to allow optimal diffusion of oxygen and substrates [13]. The dose of glucagon used in this study was half of that used in the previous study, since plasma levels of glucagon were higher than reported in stressed humans after trauma and sepsis. Plasma levels of the hormone were lower in the current study than in our previous report. Glucagon levels in the current control rats were similar to plasma concentrations reported in normal rats [25]. A twofold to threefold increase of glucagon levels during hormone infusion as observed here is in line with results in triple-hormone infused humans [11]. In a previous study, the plasma glucagon concentration was 142 pg/mL in control patients and 282 pg/mL in septic patients [26]. The most important difference between the current study and our previous report [14] was that measurements of muscle protein turnover rates were included here. The effect of catabolic hormone infusion on muscle protein synthesis and degradation rates has not been reported previously. Protein breakdown was assessed as release of tyrosine from incubated muscles in the present study. Although tyrosine release adequately reflects total protein breakdown, it does not distinguish between the degradation of myofibrillar and nonmyofibrillar proteins. We recently found that sepsis mainly stimulated myofibrillar protein breakdown, measured as release of 3-methylhistidine by incubated skeletal muscles [27]. The effect of the catabolic hormones on myofibrillar protein degradation remains to be determined. It should also be noted that the response to the catabolic hormones was determined only in the slow, red soleus muscle. The reason for this was 298
that we wanted to study a muscle in which both protein turnover and amino acid transport are substantially altered 16 hours after cecal ligation and puncture in rats [4,5]. However, although amino acid uptake was only affected by sepsis to a small extent in the white, fast extensor digitorum longus muscle [5], the increase in proteolysis was more pronounced in white than in red muscle [27]. Thus, in future experiments, the effect of catabolic hormone infusion on fast, white muscle needs to be elucidated, at least regarding protein turnover rates. Only the combined effect of the catabolic hormones was studied in the current report, and, consequently, it is not known which of the three hormones was most important for the increased muscle proteolysis. The hormones were administered simultaneously since the purpose of this study was to determine if the hormonal environment that is characteristic of injury and sepsis (i.e., increased plasma levels of all three hormones) would simulate the metabolic changes seen in sepsis. In addition to catabolic hormones, monokines, in particular interleukin- 1 (IL-1), have been suggested as regulators of muscle proteolysis during sepsis. Studies by Clowes et al [2] demonstrated the presence of a circulating factor in plasma from septic patients that enhanced muscle protein degradation, and data suggested that this factor was a cleavage product of IL- 1. Subsequent experiments from the same laboratory implied a direct stimulatory effect of IL-I~ on muscle protein breakdown [28]. Other reports, however, have questioned the role of IL- 1 in stimulated muscle proteolysis [29-31]. Studies in isolated hepatocytes suggested that an interaction between glucocorticoids and IL- 1 may be important for the metabolic response to sepsis [32]. It is possible that the smaller increase in tyrosine release from incubated soleus muscles following infusion of catabolic hormones for 16 hours compared with the increase observed 16 hours after CLP in rats [4,22] was due to a lack of co-factors required for the full metabolic response to catabolic hormones during sepsis. Although accelerated protein breakdown is probably the most important factor for muscle catabolism during sepsis, reduced protein synthesis may contribute as well. In previous reports, we found that while total protein breakdown was increased by 40% to 70% 16 hours after CLP in rats, protein synthesis was reduced by approximately 20% [4,22]. The current results suggest that re-
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duction of muscle protein synthesis is not mediated by the catabolic hormones. Other studies implied that monokines also are probably not involved in the reduction of muscle protein synthesis; since addition in vitro to incubated mouse extensor digitorum longus muscles of supernatant from stimulated monocytes or of the individual monokines I L - l a , IL-1/3, and tumor necrosis factor did not affect protein synthesis rates [30]. A possible role of thyroid hormone in reduced muscle protein synthesis in sepsis was suggested recently [22], but inhibited amino acid uptake [5,6] and local hypoperfusion and energy deficiency m a y also be considered as possible mechanisms of decreased protein synthesis. In a previous report from this laboratory [6], addition in vitro of the catabolic hormones to incubated rat skeletal muscles inhibited amino acid uptake, similar to that seen in sepsis [5,6]. In a subsequent study, however, we found that administration of the catabolic hormones in vivo stimulated amino acid uptake [14], which is similar to the present results. Thus, it is not likely that the catabolic hormones mediate the effect of sepsis on muscle amino acid transport. Recent studies from our laboratory suggest that a tow molecular weight monokine circulating in the blood during sepsis m a y account for inhibition of muscle amino acid transport [6,29]. In conclusion, the current study suggests that increased muscle proteolysis in sepsis and severe injury m a y be mediated in part by catabolic hormones. In contrast, reduced muscle protein synthesis and amino acid uptake are probably signaled by other substances or mechanisms.
These studies are fascinating in that they demonstrate the effect o f the catabolic hormones on proteolysis in severe injury. The data also show that the reduced protein synthesis and amino acid uptake do not result from simple hormone infusion.
REFERENCES 1. O'Donnell TF, Clowes GHA, Blackburn GL, Ryan NT, Benotti PN, Miller JDB. Proteolysis associated with a deficit of peripheral energy fuel substrates in septic man. Surgery 1976; 80: 192-200. 2. Clowes GHA, George BC, Villee CA, Saravis CA. Muscle proteolysis induced by a circulating peptide in patients with sepsis or trauma. N Engl J Med 1983; 308: 545-52. 3. Hasselgren PO, Jagenburg R, Karlstr6m L, Pedersen P, Seeman T. Changes of protein metabolism in liver and skeletal muscle following trauma complicated by sepsis. J Trauma 1984; 24: 224-8. 4. Hasselgren PO, Talamini MA, James JH, Fischer JE. Protein metabolism in different types of skeletal muscle during early and late sepsis in rats. Arch Surg 1986; 121: 918-23. 5. Hasselgren PO, James JH, Fischer JE. Inhibited muscle amino acid uptake in sepsis. Ann Surg 1986; 203: 360-5. 6. Hasselgren PO, James JH, Warner BW, Ogle C, Takehara H, Fischer JE. Reduced muscle amino acid uptake in sepsis and the effects invitro of septic plasma and interleukin-1. Surgery 1986; 100: 222-8. 7. Wilmore DW, Lindsey CA, Moylan JA, Faloona GR, Pruitt BA, Unger RH. Hyperglucagonaemia after burns. Lancet 1974; 1: 73-5.
8. Vaughan GM, Becker RA, Allen JP, Goodwin CW, Pruitt BA, Mason AD. Cortisol and corticotrophin in burned patients. J Trauma 1982; 22: 263-73. 9. Davies CL, Newman R J, Molyneux SG, Grahame-Smith DG. The relationship between plasma catecholamines and severity of injury in man. J Trauma 1984; 24: 99-105. 10. Shamoon HR, Hendler R, Sherwin RS. Synergistic interactions among anti-insulin hormones in the pathogenesis of stress hyperglycemia in humans. J Clin Endocrinol Metab 1981; 52: 1235-41. 11. Bessey PQ, Watters JM, Aoki TT, Wilmore DW. Combined hormone infusion simulates the metabolic response to injury. Ann Surg 1984; 200: 264-81. 12. Gelfand RA, Matthews DE, Bier DM, Sherwin RS. Role of counterregulatory hormones in the catabolic response to stress. J Clin Invest 1984; 74: 2238-48. 13. Goldberg AL, Martel SB, Kushmerick MJ. In vitro preparations of the diaphragm and other skeletal muscles. In: Hardman JG, O'Malley BW, eds. Methods in enzymology. Vol. 39. New York: Academic Press, 1975: 82-94, 14. Warner BW, James JH, Hasselgren PO, LaFrance R, Fischer JE. Effect of catabolic hormone infusion on organ amino acid uptake. J Surg Res 1987; 42: 418-24. 15. Sanders RB, Riggs TR. Modification by insulin of the distribution of two model amino acids in the rat. Endocrinology 1967; 80: 20-37, 16. Shotwell MA, Kilberg MS, Oxender DL. The regulation of neutral amino acid transport in mammalian cells. Biochim Biophys Acta 1983; 737: 267-84. 17. Shotwell MA, Lobaton CD, Collarini EJ, Moreno A, Giles RE, Oxender DL. Genetic studies of leucine transport in mammalian cells. Fed Proc 1984; 43: 2269-72. 18. Warner BW, James JH, Hasselgren PO, Hummel RP, Fischer JE. Effect of sepsis and starvation on amino acid uptake in skeletal muscle. J Surg Res 1987; 42: 377-82. 19. Rannels DE, Wartell SA, Watkins CA. The measurement of protein synthesis in biological systems. Life Sci 1982; 30:1679-90. 20. Clark AS, Mitch WE. Comparison of protein synthesis and degradation in incubated and perfused muscle. Biochem J 1983; 212: 649-53. 21. Li JB, Gotdberg AL. Effect of food deprivation on protein synthesis and degradation in rat skeletal muscles. Am J Physiol 1976; 231: 441-8. 2 2 . Hasselgren PO, Chen IW, James JH, Sperling M, Warner BW, Fischer JE. Studies on the possible role of thyroid hormone in altered muscle protein turnover during sepsis. Ann Surg !987; 206: 1 8 - 24. 23. Wilmore DW. Alterations in protein, carbohydrate, and fat metabolism in injured and septic patients. J Am Coll Nutr 1983; 2: 3-13. 24. Niggli E, RiidisiiliA, Maurer P, Weingart R. Effects of general anesthetics on current flow across membranes in guinea pig myocytes. Am J Physiol 1989; 256: C273-81. 25. Mlekusch W, Paletta B, Truppe W, et al. Plasma concentr ations of glucose, corticosterone, glucagon and insulin and liver con, tent of metabolic substrates and enzymes during starvation and additional hypoxia in the rat. Horm Metab Res 1981; 13: 612-4. 26. Marchuk JB, Finley R J, Groves AC, et al. Catabolic hormones and substrate patterns in septic patients. J Surg Res 1977; 23:17782. 27. Hasselgren PO, James JH, Benson DW, et al. Total and myofibrillar protein breakdown in different types of rat skeletal muscle: effects of sepsis and regulation by insulin. Metabolism 1989; 38: 634-40. 28. Clowes GHA, George BC, Bosari S, Love W. Induction of muscle protein degradation by recombinant interleukin-1 (rlL-1) and its spontaneously occurring fragments (abstr). J Leukocyte Biol 1987; 42: 547-8. 29. Hummel RP, Warner BW, Pedersen P, et al. In vitro effects of
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TNF, IL-la, and other monokines on skeletal muscle amino acid uptake and protein degradation. Surg Forum 1987; 38: 13-5. 30. Moldawer LL, Svaninger G, Gelin J, Lundholm KG. lnterleukin-1 and tumor necrosis factor do not regulate protein balance in skeletal muscle. Am J Physiol 1987; 253: C766-73. 31. Goldberg AL, Kettelhut IC, Furono K, Fagan JM, Baracos V. Activation of protein breakdown and prostaglandin E2 production
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in rat skeletal muscle in fever is signaled by a macrophage product distinct from interleukin-1 or other known monokines. J Clin Invest 1988; 81: 1378-83. 32. Koj A, Gauldie J, Regoeczi E, Sauder DN, Sweeney GD. The acute-phase response of cultured rat hepatocytes: system characterization and the effect of human cytokines. Biochem J 1984; 224: 505-14.
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Increased plasma levels of the catabolic hormones glucagon, epinephrine, and cortisol have been implicated in mediating various metabolic alterations in trauma and sepsis. Their role in altered protein turnover and amino acid transport in skeletal muscle during sepsis, however, is not known. In the current study, rats were infused with a mixture of the catabolic hormones for 16 hours. Control animals were infused with vehicle solution. Protein synthesis and degradation rates were measured in incubated, intact soleus muscles as incorporation of 14C-phenylalanine into protein and release of tyrosine into incubation medium, respectively. Muscle amino acid uptake was determined by measuring the intracellular to extracellular ratio of [3H]-c~-aminoisobutyric acid after incubation for 2 hours. Infusion of catabolic hormones for 16 hours resulted in elevated plasma glucose and lactate levels, reduced plasma concentrations of most amino acids, and accelerated muscle protein breakdown, similar to previous findings in septic rats. Protein synthesis rates and amino acid uptake in incubated muscles were not significantly different in control and hormoneinfused rats. The current study suggests that increased muscle proteolysis in sepsis and severe injury may be mediated in part by catabolic hormones. In contrast, reduced muscle protein synthesis and amino acid uptake are probably signaled by other substances or mechanisms.
From the Department of Surgery, Universityof Cincinnati Medical Center, Cincinnati, Ohio. Supportedin part by grant 1R01 DK3790801 fromthe National Institutes of Health, Bethesda,Maryland. Requests for reprintsshould be addressedto JosefE. Fischer,MD, Department of Surgery,Universityof Cincinnati Medical Center, 231 BethesdaAvenue(ML# 558), Cincinnati,Ohio45267. Manuscript submittedAugust 23, 1988,revisedMarch 18, 1989, and acceptedApril 5, 1989.
evere trauma and sepsis are characterized by accelerated degradation and reduced synthesis of skeletal muscle protein [1-4] and by inhibition of muscle amino acid uptake [5,6]. The mediators of these metabolic responses to sepsis are currently unknown. Plasma levels of the "catabolic hormones" glucagon, epinephrine, and cortisol are elevated after severe injury or sepsis [7-9], and administration of these hormones to normal subjects results in metabolic alterations commonly observed in stressed patients, such as hypermetabolism, negative nitrogen balance, hyperglycemia, and peripheral insulin resistance [10-12]. Consequently, the catabolic hormones have been implicated in mediating various metabolic alterations in trauma and sepsis, but their role in altered muscle protein turnover and amino acid transport is not known. The purpose of the current study was to determine the effect of catabolic hormone infusion in rats on protein synthesis and degradation and amino acid uptake in skeletal muscle.
S
MATERIAL AND M E T H O D S Male Sprague-Dawley rats weighing approximately 60 g were used. Animals of this size possess thin soleus muscles suitable for diffusion of nutrients and oxygen during in vitro incubation [13]. After acclimatization for 2 to 3 days in a room with a 12-hour light/12-hour dark cycle (240C), rats underwent central venous catheterization. With rats under pentobarbital anesthesia (45 mg/kg intraperitoneally), the neck and back regions were shaved and scrubbed with a povidone-iodine solution. An external jugular vein was cannulated with Silastic tubing (0.025-inch inner diameter), which was passed subcutaneously to exit the mid-scapular region of the back. The catheter was protected externally by a coiled steel spring attached to a swivel apparatus. After catheterization, animals were housed in individual metabolic cages and were infused with catabolic hormones or vehicle solution for 16 hours. Water, but no food, was provided during the period of hormone or control infusion. At the end of the infusion, soleus muscles were removed for studies of protein turnover and amino acid uptake. Blood was obtained by heart puncture for determination of levels of plasma amino acids, glucose, lactate, and blood urea nitrogen. Care and handling of animals in all experiments conformed to the guidelines established by the Department of Laboratory Animal Medicine of the University of Cincinnati Medical Center. The current experimental protocol involves anesthesia, surgical incision of the neck, and 16-hour fasting. Although control and hormone-infused rats underwent
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identical treatment, it is possible that the different aforemtioned factors could alter basal protein turnover rates or amino acid uptake, which in turn could alter the response to hormone infusion. Control experiments were therefore performed in which muscle protein synthesis and degradation rates and amino acid uptake were measured in fed and 16-hour fasted rats and in rats that had been anesthetized (pentobarbita145 mg/kg) or subjected to skin incision of the neck 16 hours earlier. Rats were infused with a mixture of glucagon (2.5 gg/ kg/hour), epinephrine (6 tzg/kg/hour; adrenalin chloride), and corticosterone (4.2 mg/kg/hour) dissolved in normal saline and with the addition of ascorbic acid (1 mg/mL) and albumin (3 mg/mL). Control animals received an infusion of the same solution, but without hormones. Infusions were administered at a rate of 1.1 m L / hour for 16 hours. With the exception of glucagon, the amounts of hormone administered were the same as in a previous study from our laboratory in which the infusion resulted in plasma levels consistent with those reported in injured and septic patients [14]. In that study, plasma glucagon levels were higher than previously reported after injury and sepsis. In this study, therefore, the dose of glucagon administered was half of that administered in our previous report. In separate experiments, plasma levels of glucagon were determined [14] at the end of the infusion period. Animals in these experiments were somewhat larger (body weight approximately 120 g) than in the experiments in which muscles were incubated in vitro. Since the concentrations of corticosterone and epinephrine in the current infusion solution were identical to those in our previous report [ 14], measurements of plasma concentrations of these hormones were not repeated here. Amino acid uptake was determined in incubated soleus muscles by measuring the uptake of [JH]-a-aminoisobutyric acid (A1B), a synthetic amino acid analogue that is neither metabolized nor incorporated into protein [15] and is predominantly transported by system A [16,17]. With rats under light ether anesthesia, soleus muscles were removed with intact tendons and placed individually in 25-mL Erlenmeyer flasks containing 3 mL of oxygenated (O2:CO2 = 95:5) Krebs-Henseleit bicarbonate buffer (pH 7.4) with glucose (10 mmol/L), [JH]AIB (0.5/~Ci/mL, 0.5 mmol/L), and [~4C]-inulin (0.05 ~Ci/mL). The medium was gassed with O2:CO2 (95:5), the flask was stoppered, and the incubation was performed at 37~ for 2 hours in a shaking water bath. After incubation, muscles were blotted, weighed, and dissolved in 1 mL of tissue solubilizer and 0.1 mL of water. Radioactivity of dissolved muscles and media samples was measured in a scintillation spectrophotometer. Intracellular [3H]-AIB was determined from total tissue [JH]AIB corrected for [JH]-AIB in the extracellular volume (measured as [14C].inulin space) as described previously [18]. Total tissue water of incubated soleus muscles was determined in separate experiments from wet and dry weights and was 79%. The AIB uptake was expressed as the intracellular to extracellular ratio of [JH]-AIB. For the study of protein synthesis, the muscle tendons ")96
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were tied to stainless steel frames to approximate resting length and preincubated for 30 minutes in 3 mL of oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4) with glucose (10 mm01/L). The medium was gassed with O2:CO2 (95:5), the flask was stoppered, and the incubation was performed at 37~ in a shaking water bath. After preincubation, muscles were gently blotted and transferred to 3 mL of fresh medium of the same composition as described earlier, but which also contained [14C]-phenylalanine (0.05/zCi/mL; 0.5 mmol/L). After incubation for 2 hours, the muscles were homogenized in 10% trichloroacetic acid (TCA). The precipitate was washed twice with 10% TCA and once with ether-ethanol (1:1). The pellet was dried overnight at room temperature and dissolved in 0.1 mL of water and 1 mL of tissue solubilizer, and the radioactivity was measured. Since a high concentration of phenylalanine was used in the incubation medium, the extraceUular specific radioactivity of [~4C]-phenylalanine was used to calculate amino acid incorporation into protein [19,20]. For the study of protein degradation, the Krebs-Henseleit bicarbonate buffer was supplemented with glucose (10 mmol/L), albumin (2 g/L), and physiologic levels of amino acids (with the exception of tyrosine). Soleus muscles were stretched to resting length as described earlier, and after 30 minutes, preincubation muscles were transferred to fresh medium containing cycloheximide (0.5 mmol/L) in addition to the aforementioned supplements. Proteolytic rate was assessed as release of tyrosine into the incubation medium over 2 hours as previously described [4,21]. Levels of plasma glucose and lactate were determined using a YSI Model 23A glucose and lactate analyzer, and blood urea nitrogen was measured calorimetrically. Plasma amino acids were analyzed using a 121-MB amino acid analyzer. Results are presented as mean values 4- SEM. Student's t test or analysis of variance (ANOVA), followed by Tukey's test, was used when statistical comparisons were made. RESULTS All animals tolerated the 16-hour infusion well, and there was no mortality in control or hormone-infused rats. Infusion of catabolic hormones for 16 hours resulted in significant elevations of plasma glucose and lactate levels, whereas no difference in blood urea nitrogen was noted (Table I). At the end of the infusion period, the plasma concentration of glucagon was 104 4- 3 pg/mL in control (n = 5) and 238 4- 20 pg/mL in hormone-infused rats (n --- 5; p <0.01). The plasma concentrations of all nonessential amino acids, with the exception of glutamic acid, were reduced in hormone-infused rats (Table II). Of the essential amino acids, threonine, methionine, isoleucine, lysine, histidine, and arginine were reduced. In a previous study, plasma concentrations of most amino acids were reduced in septic rats as well, although the concentrations of some amino acids were increased, most notably those of phenylalanine, tyrosine, and histidine [4].
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TABLE I Plasma Glucose and Lactate Levels and BUN In Rats Receiving Catabolic Hormone or Control Infusion for 16 Hours
Glucose (mg/dL) Lactate (mg/dL) BUN (mg/dL)
Control (n = 7)
Catabolic Hormones (n = 7)
101 4- 2 26 4- 2 14 4- 1
199 4- 4* 33 4- 2* 17 4- 2
9 p <0.01 versus odntrol. BUN = blood urea nitrogen.
AIB uptake, expressed as the intracellular to extracellular distribution ratio, was higher in soleus muscles of catabolic hormone-infused rats than in muscles of control animals, but the difference between the two groups of animals was not statistically significant (Table III). Protein synthesis rates were almost identical in incubated soleus muscles from control and hormone-infused rats, whereas protein degradation, measured as release of tyrosine, was increased by approximately 20% in the hormone-treated animals (Table liD, In control experiments, no significant differences in AIB uptake or protein synthesis or degradation rates were noted between fed rats and rats that had been subjected to a 16-hour fast, anesthesia only, or skin incision of the neck (Table IV). However, there was a significant difference in AIB uptake between 16-hour fasted rats and rats that had been subjected to anesthesia (p <0.05 by ANOVA) (Table IV). COMMENTS The current study showed that infusion of the catabolic hormones glucagon, epinephrine, and corticosterone in rats for 16 hours stimulated muscle proteolysis, measured as release of tyrosine from incubated soleus muscles. In previous reports from our laboratory, tyrosine release from incubated soleus muscles was increased 16 hours after induction of sepsis in rats [4,22], Thus, in this respect, the metabolic response to catabolic hormone infusion was similar to the metabolic response to sepsis, although the increase in tyrosine release was more pronounced in septic than in hormone-infused animals, In contrast, changes in muscle protein synthesis and amino acid uptake observed in septic rats [4-6,22] were not duplicated by hormone infusion. Thus, it is possible that some, but not all, metabolic alterations in sepsis are partially mediated by the catabolic hormones. The current finding of increased plasma levels of glucose and lactate further supports the concept that catabolic hormones play a role in metabolic alterations in sepsis [4,23]. In previous studies, triple hormone infusion in humans [10-12] and in rats [14] resulted in increased urinary nitrogen excretion. The results of the current study indicate that negative nitrogen balance reflected muscle protein breakdown, although altered protein turnover rates in other organs and tissues may have contributed.
TABLE II Plasma Amino Acid Levels ( / ~ m o l / L ) In Rats Receiving Catabolic Hormone or Control Infusion for 16 Hours Control (n = 7) Essential Threonine Valine Methionine Isoleucine Leucine Phenylalanine Tryptophan Lysine
Histidine Arglnine Nonessential Aspartlc acid Hydroxyproline Serine Asparagine Glutamic acid Glutamine Proline Glycine Alanine Tyrosine
Catabolic Hormones (n = 7)
264 250 59 132 199 78 72 377 81 127
4444444444-
7 12 1 4 4 3 7 14 1 9
124 229 31 110 179 74 71 232 67 69
4- 81. 4- 15 4- 1T 4- 6* 4- 10 4- 2 4- 7 4- 221 4- 3 T 4- 51.
25 39 289 94 56 685 173 574 324 54
4444444444-
1 3 10 4 16 28 11 42 13 2
18 14 131 62 53 428 65 213 167 37
4- 11. 4- 11 4- 41 4- 3 T 4- 4 4- 251 4- 51 4- 10 t 4- 121 4- 2 T
* p <0.05 versus control. t p <0.01 versus control.
TABLE I I I Protein Degradation, Prcteln Synthesis, and AIB Uptake In Incubated Soleus Muscles from Rats Receiving Catabolic Hormone or Control Infusion for 16 Hours
AIB uptake (intracellular/extracellular distribution ratio) Protein synthesis (nmol Phe/g 9 2h) Protein degradation (nmol Tyr/g 9 2h)
Control (n = 7)
Catabolic Hormones (n = 7)
3.86 4- 0.63
5.16 4- 0.56
170 4- 20
170 4- 10
370 4- 10
440 4- 20*
* p <0.02 versus control. Phe -- phenylalanine; Tyr = tyrosine.
It should be noted that the present experimental protocol included anesthesia, skin incision of the neck for placement of a venous catheter, and a 16-hour fasting period. Although both control and hormone-infused rats were treated identically, it may be argued that anesthesia, surgical incision, or fasting could alter basal AIB uptake and protein turnover rates, thereby influencing the response to hormone infusion. Control experiments, however, did not support that concept, since anesthesia alone, skin incision, or a 16-hour fast did not result in significant changes compared with fed, untreated rats. We do not currently have an explanation for the high AIB uptake in animals that had been subjected to anesthesia only, but a
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TABLE IV A I B U p t a k e and Protein Synthesis and D e g r a d a t i o n in I n c u b a t e d Soleus Muscles 16-Hour Fasted
Fed Rats AIB uptake (intracellular/extracellular Protein synthesis (nmol Phe/g ~ 2h)
distribution ratio)
Protein degradation (nrnol Tyr/g 9 2h)
Rats
Anesthesia
(n=6)
(n = 6 )
(n=6)
6.03 4- 0.65 292 4- 15 202 4- 29
4.79 4- 0.29 225 4- 18 250 4- 33
8.14 4- 0.42" 250 -4- 33 266 4- 18
Neck Incision (n=6)
6,72 4- 0.29 259 4- 5 270 4- 7
9 p<0.05 versus 16-hour fasted rats by ANOVA. Phe = phenylalanine; Tyr = tyrosine.
recent study suggests that general anesthetics can affect muscle cell membrane functions [24]. We recently reported another study in which catabolic hormones were infused in rats [14]. That study, however, differs from the current report in several important aspects. In this study, animals were infused for 16 hours since altered protein turnover rates and amino acid uptake were observed 16 hours after induction of sepsis in rats [4,22]. In contrast, in our previous report, animals were infused for 72 hours [14], similar to human studies reported by Bessey et al [II]. The animals used in this study were smaller (approximately 60 g) than those used in our previous report (approximately 100 to 150 g) and muscles were studied in vitro, whereas AIB uptake was measured in vivo in our earlier paper [14]. Small muscles are required for in vitro incubations to allow optimal diffusion of oxygen and substrates [13]. The dose of glucagon used in this study was half of that used in the previous study, since plasma levels of glucagon were higher than reported in stressed humans after trauma and sepsis. Plasma levels of the hormone were lower in the current study than in our previous report. Glucagon levels in the current control rats were similar to plasma concentrations reported in normal rats [25]. A twofold to threefold increase of glucagon levels during hormone infusion as observed here is in line with results in triple-hormone infused humans [11]. In a previous study, the plasma glucagon concentration was 142 pg/mL in control patients and 282 pg/mL in septic patients [26]. The most important difference between the current study and our previous report [14] was that measurements of muscle protein turnover rates were included here. The effect of catabolic hormone infusion on muscle protein synthesis and degradation rates has not been reported previously. Protein breakdown was assessed as release of tyrosine from incubated muscles in the present study. Although tyrosine release adequately reflects total protein breakdown, it does not distinguish between the degradation of myofibrillar and nonmyofibrillar proteins. We recently found that sepsis mainly stimulated myofibrillar protein breakdown, measured as release of 3-methylhistidine by incubated skeletal muscles [27]. The effect of the catabolic hormones on myofibrillar protein degradation remains to be determined. It should also be noted that the response to the catabolic hormones was determined only in the slow, red soleus muscle. The reason for this was 298
that we wanted to study a muscle in which both protein turnover and amino acid transport are substantially altered 16 hours after cecal ligation and puncture in rats [4,5]. However, although amino acid uptake was only affected by sepsis to a small extent in the white, fast extensor digitorum longus muscle [5], the increase in proteolysis was more pronounced in white than in red muscle [27]. Thus, in future experiments, the effect of catabolic hormone infusion on fast, white muscle needs to be elucidated, at least regarding protein turnover rates. Only the combined effect of the catabolic hormones was studied in the current report, and, consequently, it is not known which of the three hormones was most important for the increased muscle proteolysis. The hormones were administered simultaneously since the purpose of this study was to determine if the hormonal environment that is characteristic of injury and sepsis (i.e., increased plasma levels of all three hormones) would simulate the metabolic changes seen in sepsis. In addition to catabolic hormones, monokines, in particular interleukin- 1 (IL-1), have been suggested as regulators of muscle proteolysis during sepsis. Studies by Clowes et al [2] demonstrated the presence of a circulating factor in plasma from septic patients that enhanced muscle protein degradation, and data suggested that this factor was a cleavage product of IL- 1. Subsequent experiments from the same laboratory implied a direct stimulatory effect of IL-I~ on muscle protein breakdown [28]. Other reports, however, have questioned the role of IL- 1 in stimulated muscle proteolysis [29-31]. Studies in isolated hepatocytes suggested that an interaction between glucocorticoids and IL- 1 may be important for the metabolic response to sepsis [32]. It is possible that the smaller increase in tyrosine release from incubated soleus muscles following infusion of catabolic hormones for 16 hours compared with the increase observed 16 hours after CLP in rats [4,22] was due to a lack of co-factors required for the full metabolic response to catabolic hormones during sepsis. Although accelerated protein breakdown is probably the most important factor for muscle catabolism during sepsis, reduced protein synthesis may contribute as well. In previous reports, we found that while total protein breakdown was increased by 40% to 70% 16 hours after CLP in rats, protein synthesis was reduced by approximately 20% [4,22]. The current results suggest that re-
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duction of muscle protein synthesis is not mediated by the catabolic hormones. Other studies implied that monokines also are probably not involved in the reduction of muscle protein synthesis; since addition in vitro to incubated mouse extensor digitorum longus muscles of supernatant from stimulated monocytes or of the individual monokines I L - l a , IL-1/3, and tumor necrosis factor did not affect protein synthesis rates [30]. A possible role of thyroid hormone in reduced muscle protein synthesis in sepsis was suggested recently [22], but inhibited amino acid uptake [5,6] and local hypoperfusion and energy deficiency m a y also be considered as possible mechanisms of decreased protein synthesis. In a previous report from this laboratory [6], addition in vitro of the catabolic hormones to incubated rat skeletal muscles inhibited amino acid uptake, similar to that seen in sepsis [5,6]. In a subsequent study, however, we found that administration of the catabolic hormones in vivo stimulated amino acid uptake [14], which is similar to the present results. Thus, it is not likely that the catabolic hormones mediate the effect of sepsis on muscle amino acid transport. Recent studies from our laboratory suggest that a tow molecular weight monokine circulating in the blood during sepsis m a y account for inhibition of muscle amino acid transport [6,29]. In conclusion, the current study suggests that increased muscle proteolysis in sepsis and severe injury m a y be mediated in part by catabolic hormones. In contrast, reduced muscle protein synthesis and amino acid uptake are probably signaled by other substances or mechanisms.
These studies are fascinating in that they demonstrate the effect o f the catabolic hormones on proteolysis in severe injury. The data also show that the reduced protein synthesis and amino acid uptake do not result from simple hormone infusion.
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8. Vaughan GM, Becker RA, Allen JP, Goodwin CW, Pruitt BA, Mason AD. Cortisol and corticotrophin in burned patients. J Trauma 1982; 22: 263-73. 9. Davies CL, Newman R J, Molyneux SG, Grahame-Smith DG. The relationship between plasma catecholamines and severity of injury in man. J Trauma 1984; 24: 99-105. 10. Shamoon HR, Hendler R, Sherwin RS. Synergistic interactions among anti-insulin hormones in the pathogenesis of stress hyperglycemia in humans. J Clin Endocrinol Metab 1981; 52: 1235-41. 11. Bessey PQ, Watters JM, Aoki TT, Wilmore DW. Combined hormone infusion simulates the metabolic response to injury. Ann Surg 1984; 200: 264-81. 12. Gelfand RA, Matthews DE, Bier DM, Sherwin RS. Role of counterregulatory hormones in the catabolic response to stress. J Clin Invest 1984; 74: 2238-48. 13. Goldberg AL, Martel SB, Kushmerick MJ. In vitro preparations of the diaphragm and other skeletal muscles. In: Hardman JG, O'Malley BW, eds. Methods in enzymology. Vol. 39. New York: Academic Press, 1975: 82-94, 14. Warner BW, James JH, Hasselgren PO, LaFrance R, Fischer JE. Effect of catabolic hormone infusion on organ amino acid uptake. J Surg Res 1987; 42: 418-24. 15. Sanders RB, Riggs TR. Modification by insulin of the distribution of two model amino acids in the rat. Endocrinology 1967; 80: 20-37, 16. Shotwell MA, Kilberg MS, Oxender DL. The regulation of neutral amino acid transport in mammalian cells. Biochim Biophys Acta 1983; 737: 267-84. 17. Shotwell MA, Lobaton CD, Collarini EJ, Moreno A, Giles RE, Oxender DL. Genetic studies of leucine transport in mammalian cells. Fed Proc 1984; 43: 2269-72. 18. Warner BW, James JH, Hasselgren PO, Hummel RP, Fischer JE. Effect of sepsis and starvation on amino acid uptake in skeletal muscle. J Surg Res 1987; 42: 377-82. 19. Rannels DE, Wartell SA, Watkins CA. The measurement of protein synthesis in biological systems. Life Sci 1982; 30:1679-90. 20. Clark AS, Mitch WE. Comparison of protein synthesis and degradation in incubated and perfused muscle. Biochem J 1983; 212: 649-53. 21. Li JB, Gotdberg AL. Effect of food deprivation on protein synthesis and degradation in rat skeletal muscles. Am J Physiol 1976; 231: 441-8. 2 2 . Hasselgren PO, Chen IW, James JH, Sperling M, Warner BW, Fischer JE. Studies on the possible role of thyroid hormone in altered muscle protein turnover during sepsis. Ann Surg !987; 206: 1 8 - 24. 23. Wilmore DW. Alterations in protein, carbohydrate, and fat metabolism in injured and septic patients. J Am Coll Nutr 1983; 2: 3-13. 24. Niggli E, RiidisiiliA, Maurer P, Weingart R. Effects of general anesthetics on current flow across membranes in guinea pig myocytes. Am J Physiol 1989; 256: C273-81. 25. Mlekusch W, Paletta B, Truppe W, et al. Plasma concentr ations of glucose, corticosterone, glucagon and insulin and liver con, tent of metabolic substrates and enzymes during starvation and additional hypoxia in the rat. Horm Metab Res 1981; 13: 612-4. 26. Marchuk JB, Finley R J, Groves AC, et al. Catabolic hormones and substrate patterns in septic patients. J Surg Res 1977; 23:17782. 27. Hasselgren PO, James JH, Benson DW, et al. Total and myofibrillar protein breakdown in different types of rat skeletal muscle: effects of sepsis and regulation by insulin. Metabolism 1989; 38: 634-40. 28. Clowes GHA, George BC, Bosari S, Love W. Induction of muscle protein degradation by recombinant interleukin-1 (rlL-1) and its spontaneously occurring fragments (abstr). J Leukocyte Biol 1987; 42: 547-8. 29. Hummel RP, Warner BW, Pedersen P, et al. In vitro effects of
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TNF, IL-la, and other monokines on skeletal muscle amino acid uptake and protein degradation. Surg Forum 1987; 38: 13-5. 30. Moldawer LL, Svaninger G, Gelin J, Lundholm KG. lnterleukin-1 and tumor necrosis factor do not regulate protein balance in skeletal muscle. Am J Physiol 1987; 253: C766-73. 31. Goldberg AL, Kettelhut IC, Furono K, Fagan JM, Baracos V. Activation of protein breakdown and prostaglandin E2 production
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