Metabolism Clinica 2 and Experimental NOVEMBER
VOL 39, NO 11
Protein
Synthesis in Skeletal Muscle During Starvation and Refeeding: of Data From Intact Muscle and Muscle Biopsy Material Kristina Magnusson,
John Wahren,
1990
Comparison
and Lars Ekman
The intact extensor digitorum longus (EDL) preparation in rat is a well-documented model for assessing protein synthesis in skeletal muscle. Human muscle biopsy material has also been used, but the extent to which biopsy material is representative for evaluation of muscle protein synthesis has not been established. Therefore, the aim of this study was to compare protein synthesis in intact muscle and in muscle biopsy material simultaneously in rats. The animals (70 g) were divided into three groups: fed (n = 22). starved for 36 hours (n = 221, and refed for 24 hours (n = 19). Protein synthesis and RNA content were measured in each group. Protein synthesis was determined as the incorporation of 14-Cphenylalanine into muscle protein in the intact EDL muscle from one leg and in a muscle biopsy from the contralateral EDL muscle. The incorporation of 14C-phenylalanine was linear over time in both preparations, but was consistently lower in the muscle biopsy compared with the intact muscle. The relative change in incorporation, in % of that obtained in the fed state, showed a decrease in incorporation after 36 hours of starvation, in both intact muscle and in muscle biopsy material, 33% ? 10% and 42% + 6%. respectively. After 24 hours of refeeding. an overshoot in protein synthesis was seen. to 136% + 6% in the intact muscle and to 133% + 6% in the muscle biopsy, as compared with the fed state. The RNA content decreased during the starvation period from 21.6 + 0.7 to 14.5 + 0.4 mg RNA/g protein. However, unlike the incorporation of 14C-phenylalanine, the RNA content remained low, 14.6 t 0.5 mg RNA/g protein, after 24 hours of refeeding. In conclusion, muscle biopsy specimens and intact muscle are equally useful in the qualitative estimation of muscle protein synthesis. 0 1990 by W.B. Saunders Company.
KELETAL MUSCLE is the largest tissue in the body, constituting approximately 40% of the body weight and 75% of the lean body mass.’ It is also the largest nitrogen source in the body and accounts for approximately 50% of the whole-body protein synthesis in the overnight fasted state.’ Skeletal muscle accordingly plays an important role in whole-body protein metabolism, and even minor changes in muscle protein synthesis and/or degradation lead to changes in whole-body nitrogen economy. Protein synthesis and degradation in muscle vary markedly under different physiological and pathophysiological conditions.3-7 However, conflicting results in similar states have led to controversy regarding the reproducibility and validity of the methods used for evaluating muscle protein kinetics in mammals. The methods for evaluating protein synthesis rates in human muscle, in vivo and in vitro, have shortcomings in that they are either technically complex or based on assumptions that have not been validated.3,8 The procedure according to Fulks et al9 and Li et a14,” is based on the in vitro incorporation of amino acid into protein, using intact muscles in small animals. This technique is well characterized and
S
Metabolism,Vol39,No
ll~November),1990:pp
1113-1117
has been extensively used. ‘O-I4Results from measurements using the same procedure in muscle biopsies from large animals or humans’,” have been questioned,‘6 since the biopsy specimen may not be identical to intact muscle with regard to its ability to synthesize proteins. Consequently, the aim of this study was to validate the procedure for estimation of protein synthesis in muscle biopsies by simultaneously comparing protein synthesis in intact muscle and in a muscle biopsy from the same rat under varying nutritional conditions.
From the Vitrum Institute of Human Nuwition, Stockholm: and the Department of Clinical Physiology, the Karolinska Institute at Karolinska Hospital, Stockholm, Sweden. Supported by a fellowship grant to KM. from the European Society of Parenteral and Enteral Nutrition and the Western Nutrition Foundation Fellowship. Address reprint requests to Professor John Wahren. Department of Clinical Physiology, Karolinska Institute, Karolinska Hospital, Box 60500, S-104 01 Stockholm, Sweden. @ 1990 by W.B. Saunders Company. 00260495/90/39ll-00001$03.00/0
1113
1114
MAGNUSSON,
MATERIAL
AND METHODS
Animals Male Sprague-Dawley rats 18 days old and weighing approximately 45 to 50 g at arrival were used. They were kept in separate cages in a room with a 12-hour light-dark cycle, with constant temperature (21 to 22OC) and humidity (50%). Procedure The rats (n = 63) were conditioned for a 6-day period before the study. During this period, the animals were fed a standard laboratory diet (R3, Ewos AB, Sijdertalje, Sweden) and water, both given ad libitum. This was followed by 36 hours of starvation, when only water was given. After starvation, the animals were refed for 24 hours with pellets and water, given ad libitum. Twenty-two rats were studied in the fed state, 22 after starvation, and 19 after refeeding. In addition, animals were observed for 48 and 72 hours of refeeding, with regard to body weight. The food intake of young rats shows very small interanimal variationI and was therefore not monitored. Body weight was determined each day. Protein synthesis and RNA content of muscle tissue were measured in each group. Before the muscle preparation, animals were anaesthetized at 9:00 AM by intraperitoneal injection of pentobarbital (Mebumal, 60 mg/mL, AC0 Liikemedel, Stockholm, Sweden) at a dose of 1 mL/kg body weight. Protein Synthesis in Intact Muscle and in Muscle Biopsy Specimens The intact extensor digitorum longus muscle (EDL) from one leg was carefully dissected, without stretching the muscle, and weighed. A biopsy was taken from the contralateral EDL muscle with a biopsy nipper. The incorporation of 14Gphenylalanine into protein was determined with the same principles as described by Fulks et al9 and Li et a14.‘0using 14-C-tyrosine. The procedure followed the identical method described by Svaninger et al.” The muscle preparations were incubated in McCoy’s cell culture medium containing 1.0 pmol/L of Hepes (N-2-hydroxyethylpiperazine-N’-2-ethansulfonic acid) and U-14-C-phenylalanine (lo6 dpm/3 mL) at +37’C. Phenylalanine was chosen, since it is not metabolized by skeletal muscle and equilibration is rapid between the intracellular pool and the medium.” The medium was adjusted to pH 7.4 and saturated with a 95% 0, and 5% CO, gas mixture before incubation. The incubation was terminated after 30, 60, and 90 minutes by adding ice-chilled 5% trichloroacetic acid (TCA). The muscle preparations were washed twice with the TCA to make sure that unincorporated label was removed. Lipids were extracted sequentially by adding chloroform:methanol 1: 1, ethanol:acetone 1:1, and finally pure ether. The muscle specimens were freeze-dried and the fat-free dry weight (fdw) was determined. The muscles were dissolved in a mixture of 0.5 mL distilled water and 1.5 mL Soluene 350 and radioactivity was determined in a scintillation counter. Protein synthesis was calculated as dpm/mg fdw per hour. The specific activity in the medium was used as an approximation for intracellular specific activity, as described elsewhere.” Since it has been speculated that the specific activity between the medium and the intracellular pool may vary with different physiological states, a separate study was performed in order to evaluate if the amino acid concentration in the medium could have influenced the results in the first study. Twelve rats were divided into three groups: fed, starved for 36 hours, and refed for 24 hours. Muscle biopsies were incubated in media containing one and 10 times the plasma concentration of all amino acids. The biopsies were treated as described above.
WAHREN.
AND EKMAN
Protein content was determined” in some muscles in order to quantify the correlation between protein content and fdw. The remaining part of the EDL muscle was immediately weighed and frozen in liquid nitrogen for RNA determination according to Munro and Fleck.19 Chemicals L-(U-14Qphenylalanine 0.55 mCi/mmol was obtained from New England Nuclear, Boston, MA. McCoy cell culture medium was from Flow Laboratories, London, UK. pH was held constant with Hepes, Sigma Chemical, St Louis, MO. The muscle tissue was dissolved in Soluene 350 from Packard Instrument, Maryden, CT. All chemicals used were of highest analytical purity. Data are presented as mean + SEM for each group of rats. Standard statistical methods were employed, using Student’s t test when applicable. When data were compared on relative basis, expressed as percent of their controls, the logarithm values were used and Wilcoxon unpaired test employed. RESULTS
Body Weight The rats gained approximately 5 k 1 g/d during the conditioning period prior to the study. After 36 hours starvation, the body weight had decreased by 28% or 22 + 1 g (Fig 1). Following 24 hours of refeeding, the animals had gained 15 + 1 g or 26%. After 2 more days, the rats had gained an additional 30 * 1 g or 42%. Protein Synthesis Incorporation of 14-C-phenylalanine into muscle protein was found to be a linear function of time for at least 90 minutes in both intact muscles and muscle biopsies. This was observed in all feeding conditions (Fig 2). However, the in-
0
2
4
6
6
10
12
Body weight (g) during conditioning period. Fig 1. hours of starvation, and after 3 days of refeeding.
Days
after
36
1115
PROTEIN SYNTHESIS IN SKELETAL MUSCLE
STARVED 36h
REFED 24h
-
‘“1
Fig 2. Incorporation and refed states.
of 14-C-phenylalanine
into protein fdpm/mg
corporation of 14-C-phenylalanine was consistently lower in the muscle biopsies compared with the intact muscle (Fig 2). Protein synthesis as evaluated by the incorporation of 14-C-phenylalanine in fed rats was 609 2 32 dpm/mg freeze-dried weight (fdw) per hour in intact muscles and 268 + 22 dpm/mg fdw per hour in muscle biopsies. After 36 hours of starvation the incorporation was significantly decreased in both preparations, 200 2 21 and 113 + 7 dpm/mg fdw per hour, respectively. After 24 hours of refeeding, protein synthesis was significantly increased compared with the fed and fasted states, both in intact muscles, 831 + 46 dpm/mg fdw per hour, and in muscle biopsies, 355 + 20 dpm/mg fdw per hour (Fig 2). Since the specific aim of this study was to compare the relative change in protein synthesis in intact muscles and muscle biopsies under different feeding conditions, the values were expressed as percent of those obtained in the fed state (Fig 3). Both muscle preparations showed a significant decrease to approximately 40% of the initial value, 33% * 10% in the intact muscles and 42% f 6% in the muscle biopsies after the starvation period. After 24 hours of refeeding, an overshoot to approximately 135% was seen in both preparations, 136% t 6% and 133% + 6%, respectively (Fig 4). Thus, the relative change in protein synthesis was similarly reflected in intact muscle and in the muscle biopsy material. In the second study, the relative change in protein synthesis were similar, when muscle biopsies were incubated in medium containing one and 10 times plasma amino acid concentrations. In the medium containing one time the plasma amino acid concentration the incorporation decreased to 50% after 36 hours of starvation and increased to 126% after 24 hours of refeeding. When using an incubation medium containing 10 times the plasma amino acid concentration, the incorporation was 57% and 113%, respectively. Thus, no statistical difference was seen between the two studies.
fdw) in intact muscle (ml and in muscle biopsy
1El)in the
fed, starved,
DISCUSSION It has been suggested that muscle protein synthesis measured in vitro in muscle biopsy material may be used to assess relative changes in the rate of protein synthesis during refeeding in cancer and postoperative states. This notion has not met with general acceptance.16 The reluctance to accept muscle biopsy material for measurements of protein synthesis is in contrast to the general use of the intact EDL muscle in rats for evaluating the influence of varying nutritional and hormonal stimuli on muscle protein synthesis.“,‘2.‘4.20 Therefore, the purpose of the present study was to compare, in the same animal, measurements of protein synthesis by the two methods. Starvation and refeeding were chosen as two conditions under which the two methods may be compared,
150$ 1 100
75
50
25
0i RNA Content The RNA content was significantly decreased, from 21.6 f 0.70 mg/g protein in the nonstarved rats to 14.5 + 0.41 mg/g protein, or 33%, after 36 hours of starvation. In contrast to the increased incorporation of 14-C-phenylalanine, the RNA content remained low, 14.8 f 0.48 mg/g protein, after 24 hours refeeding (Fig 4).
FED
STARVED
REFED
36 h
24 h
Fig 3. Protein synthesis after 36 hours of starvation and 24 hours of refeeding, relative to normal feeding in intact muscle (B) and in muscle biopsy @). Asterisks denote that the difference are caused by random factors, l*P < .DDDl if compared with the fed state; lP < .Ol if compared with the fed state (Wilcoxon unpaired test).
1116
MAGNUSSON,
25
I-
i-
l-r-
FED
STARVED 36 h
REFED 24 h
RNA content in muscles in rats, after 36 and after 24 of refeeding. Values are expressed as mg RNA/g the difference are caused by random factors, l*f -C .OOOl if compared with the fed state (Student’s unpaired t test). Fig 4.
since their respective effects on protein synthesis are well documented.*’ Rats weighing between 56 and 74 g were used, because 70 g is a minimal weight for survival during 24 hours of starvation and animals in this weight range provide EDL preparations that are small enough for optimal incubation conditions.22 A 30% decrease in body weight was observed during the starvation period, corresponding to the findings shown by Li et al.4 During the same period of starvation, they showed a 15% decrease in EDL wet weight. However, the dry weight to wet weight ratio did not change, indicating a loss of nonprotein components, such as glycogen, fat, and RNA. Since the metabolic rate is approximately 10 to 20 times higher in small rodents than in man,’ the 36-hour starvation period of the present study can be compared with long-term starvation in man. Therefore, the direction of the changes in protein synthesis shows a pattern similar to that in starved and refed man.* This study demonstrates that the muscle biopsies decreased their amino acid incorporation into protein by 58% during the starvation period and increased it by 33% during refeeding, compared with the values obtained in the fed state. The corresponding changes for the intact muscle were 67% decrease during starvation and 36% increase during refeeding. This demonstrates that the two procedures for evaluating protein synthesis in muscle are equally useful for assessing relative changes in amino acid incorporation during
WAHREN, AND EKMAN
starvation and refeeding. The metabolic intervention in the animals must be regarded as substantial, as reflected in the changes in body weight and incorporation capacity. It therefore remains to be determined whether the two methods are equally capable of assessing moderate to minor changes in protein synthetic capacity. This study also provides information regarding the relationship between protein synthesis and RNA content during starvation and refeeding. In both respects there was a significant decrease after 36 hours of starvation, but during the refeeding period the trends diverged, with an increase in protein synthesis and a persistently low RNA content. It has been shown that short-term refeeding results in an acute shift in the relative polyribosomal distribution.’ The primary response to refeeding seems to be a stimulation of the activity of RNA present in the cell; secondarily new synthesis of RNA takes place. While changes in RNA content appear adequately to reflect the depression of protein synthesis during starvation, no response to short-term refeeding was observed. It remains to be determined whether, during refeeding beyond 36 hours, changes in RNA content may reflect protein synthesis. Protein synthesis in the present study was expressed as dpm/mg fdw per hour. In some muscles protein content and fdw were compared. The fdw overestimated protein content by 10% i- 1% compared with protein determination. Since the difference was constant, protein synthesis expressed as dpm/mg fdw was considered to be as precise as dpm/mg protein. 14Gphenylalanine was chosen for the incorporation measurements, since it rapidly equilibrates between the medium and the intracellular pool and is neither synthesized nor degraded in the muscle tissue.’ However, neither intact muscle nor muscle biopsy preparations show the same specific activity in the two compartments, when using an incubation medium containing plasma concentrations of all amino acids.“js” If the muscle biopsy material is incubated in a medium containing 10 times the plasma concentrations of amino acids, the specific activity between the medium and the intracellular pool becomes equal.16 In the present study, we chose an amino acid solution with concentrations of amino acids similar to those of plasma. The reason for this was that an incubation medium with supraphysiological amino acid concentrations may in itself have an impact on the protein synthesis in that, for instance, leucine is considered to exert a stimulatory effect on protein synthesis.9.20 At physiological concentrations of amino acids in the medium, the ratio between the specific activities in the medium and the intracellular pool is similar in the intact muscle and the muscle biopsy material, respectively.‘6 Therefore, changes in incorporation rate in the two preparations cannot be explained by differences in the specific activity. It has previously been suggested that the specific activity between the medium and the intracellular pool will vary with physiological states. Therefore, comparison of muscle biopsies incubated in only one time the plasma amino acid concentration could be misleading, when different physiological states are compared. This was addressed by comparing the relative incorporation rate between biopsies incubated in
PROTEIN SYNTHESIS
IN SKELETAL MUSCLE
1117
and 10 times the plasma concentrations in fed rats and after starvation and refeeding. These results indicate that the observations made at physiological concentrations cannot be explained by differences in specific activity during various nutritional states. The amino acid incorporation capacity was found to be approximately 2.3 times greater in the intact muscle compared with the muscle biopsy material. This demonstrates the quantitative importance of maintaining the integrity of the muscle for protein synthesis evaluation. However, when investigating various nutritional, hormonal, or other metabolic interventions, it is the relative change in protein synthesis rather than the absolute values that is of imporone
tance, since the relative value describes the direction and magnitude of the changes in protein synthesis regardless of the absolute incorporation of labeled amino acids. In conclusion, the present results demonstrate that muscle biopsy specimens and intact muscle are equally useful in the estimation of changes in muscle protein synthesis in response to starvation and refeeding. Further work is required to define the ability of the two methods to detect small alterations in protein synthesis. The current findings validate the use of the biopsy procedure, thereby facilitating measurements of protein synthesis in human skeletal muscle under varying clinical conditions,
REFERENCES
1. Rennie MJ: Importance of muscle to whole-body protein turnover. Br J lntraven Ther 12:3-13,198l 2. Rennie MJ, Edwards RHT, Halliday D, et al: Muscle protein synthesis measured by stable isotope techniques in man: The effects of feeding and fasting. Clin Sci 63519-523, 1982 3. Lundholm K, Edstriim S, Ekman L, et al: A comparative study of the influence of malignant tumor on the host metabolism in mice and man. Cancer 42:453-461, 1978 4. Li JB, Goldberg AL: Effects of food deprivation on protein synthesis and degradation in rat skeletal muscles. Am J Physiol 231:441-448, 1976 5. Millward DJ, Garlick PJ, Nnanyelugo DO, et al: The relative importance of muscle protein synthesis and breakdown in the regulation of muscle mass. Biochem J 156:185-188, 1976 6. Tischler ME, Fagan JM: Response to trauma of protein, amino acid and carbohydrate metabolism in injured and uninjured rat skeletal muscle. Metabolism 32:853-868, 1983 7. Stein TP, Leskiw J, Wallace HW, et al: Changes in protein synthesis after trauma: Importance of nutrition. Am J Physiol 233:E348-355, 1977 8. Morgan HE, Earl DCN, Broadus A, et al: Regulation of protein synthesis in heart muscle. I. Effect of amino acid levels on protein synthesis. J Biol Chem 246:2152-2162, 1971 9. Fulks RM, Li JB, Goldberg AL: Effect of insulin, glucose and amino acids on protein turnover in rat diaphragm. J Biol Chem 250:290-298, 1975 10. Li JB, Fulks RM, Goldberg AL: Evidence that the intracellular pool of tyrosine serves as precursor for protein synthesis in muscle. J Biol Chem 248:7272-7275, 1973 11. Svaninger G, Bennegard K, Ekman L, et al: Lack of evidence for elevated breakdown rate of skeletal muscle in weight losing tumor bearing mice. JNCI 71:341-346, 1983
12. Wernerman J, Magnusson K, Ekman L, et al: A comparison of three methods assessing protein synthesis in rat skeletal muscle. J Surg Res 43:329-336, 1987 13. Ekman L, Karlberg I, Edstrom S, et al: Metabolic alterations in liver, skeletal muscle and fat tissue in response to different tumor burdens in growing sarcoma bearing rats. J Surg Res 33:23-31,1982 14. Li JB: Protein synthesis and degradation in skeletal muscle of normal and dystrophic hamsters. Am J Physiol 239:E401-406, 1980 15. Lundholm K, Bylund A-C, Holm J, et al: Metabolic studies in human skeletal muscle tissue. Eur Surg Res 7:65-82, 1975 16. Greig PD, Rosovski SJ, Elwyn DH, et al: Protein synthesis and degradation in biopsies of rat skeletal muscle. J Surg Res 40:248-260,1986 17. Lundholm K, Schersten T: Incorporation of leucine into human skeletal muscle proteins. A study of tissue amino acid pools and their role in protein biosynthesis. Acta Physiol Stand 93:433441,197s 18. Lowry OH, Rosenbrough NJ, Farr AL, et al: Protein measurements with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 19. Munro HN, Fleck A: The determination Methods Biochem Anal 14:113-176, 1966
of nucleic
acids.
20. Lundholm K, Edstrom S, Ekman L, et al: Protein degradation in human skeletal muscle tissue: the effect of insulin, leucine, amino acids and ions. Clin Sci 60:319-326, 1981 21. Waterlow JC, Garlick PJ, Millward DJ: Protein Turnover in Mammalian Tissues and in the Whole Body. Amsterdam, The Netherlands, Elsevier/North Holland Biomedical, 1978, pp 443-479 22. Goldberg AL, Martel SB, Kushmerick MJ: In vitro preparations of the diaphragm and other skeletal muscles. Methods Enzymol39:82-93, 1975
VOL 39, NO 11
Protein
Synthesis in Skeletal Muscle During Starvation and Refeeding: of Data From Intact Muscle and Muscle Biopsy Material Kristina Magnusson,
John Wahren,
1990
Comparison
and Lars Ekman
The intact extensor digitorum longus (EDL) preparation in rat is a well-documented model for assessing protein synthesis in skeletal muscle. Human muscle biopsy material has also been used, but the extent to which biopsy material is representative for evaluation of muscle protein synthesis has not been established. Therefore, the aim of this study was to compare protein synthesis in intact muscle and in muscle biopsy material simultaneously in rats. The animals (70 g) were divided into three groups: fed (n = 22). starved for 36 hours (n = 221, and refed for 24 hours (n = 19). Protein synthesis and RNA content were measured in each group. Protein synthesis was determined as the incorporation of 14-Cphenylalanine into muscle protein in the intact EDL muscle from one leg and in a muscle biopsy from the contralateral EDL muscle. The incorporation of 14C-phenylalanine was linear over time in both preparations, but was consistently lower in the muscle biopsy compared with the intact muscle. The relative change in incorporation, in % of that obtained in the fed state, showed a decrease in incorporation after 36 hours of starvation, in both intact muscle and in muscle biopsy material, 33% ? 10% and 42% + 6%. respectively. After 24 hours of refeeding. an overshoot in protein synthesis was seen. to 136% + 6% in the intact muscle and to 133% + 6% in the muscle biopsy, as compared with the fed state. The RNA content decreased during the starvation period from 21.6 + 0.7 to 14.5 + 0.4 mg RNA/g protein. However, unlike the incorporation of 14C-phenylalanine, the RNA content remained low, 14.6 t 0.5 mg RNA/g protein, after 24 hours of refeeding. In conclusion, muscle biopsy specimens and intact muscle are equally useful in the qualitative estimation of muscle protein synthesis. 0 1990 by W.B. Saunders Company.
KELETAL MUSCLE is the largest tissue in the body, constituting approximately 40% of the body weight and 75% of the lean body mass.’ It is also the largest nitrogen source in the body and accounts for approximately 50% of the whole-body protein synthesis in the overnight fasted state.’ Skeletal muscle accordingly plays an important role in whole-body protein metabolism, and even minor changes in muscle protein synthesis and/or degradation lead to changes in whole-body nitrogen economy. Protein synthesis and degradation in muscle vary markedly under different physiological and pathophysiological conditions.3-7 However, conflicting results in similar states have led to controversy regarding the reproducibility and validity of the methods used for evaluating muscle protein kinetics in mammals. The methods for evaluating protein synthesis rates in human muscle, in vivo and in vitro, have shortcomings in that they are either technically complex or based on assumptions that have not been validated.3,8 The procedure according to Fulks et al9 and Li et a14,” is based on the in vitro incorporation of amino acid into protein, using intact muscles in small animals. This technique is well characterized and
S
Metabolism,Vol39,No
ll~November),1990:pp
1113-1117
has been extensively used. ‘O-I4Results from measurements using the same procedure in muscle biopsies from large animals or humans’,” have been questioned,‘6 since the biopsy specimen may not be identical to intact muscle with regard to its ability to synthesize proteins. Consequently, the aim of this study was to validate the procedure for estimation of protein synthesis in muscle biopsies by simultaneously comparing protein synthesis in intact muscle and in a muscle biopsy from the same rat under varying nutritional conditions.
From the Vitrum Institute of Human Nuwition, Stockholm: and the Department of Clinical Physiology, the Karolinska Institute at Karolinska Hospital, Stockholm, Sweden. Supported by a fellowship grant to KM. from the European Society of Parenteral and Enteral Nutrition and the Western Nutrition Foundation Fellowship. Address reprint requests to Professor John Wahren. Department of Clinical Physiology, Karolinska Institute, Karolinska Hospital, Box 60500, S-104 01 Stockholm, Sweden. @ 1990 by W.B. Saunders Company. 00260495/90/39ll-00001$03.00/0
1113
1114
MAGNUSSON,
MATERIAL
AND METHODS
Animals Male Sprague-Dawley rats 18 days old and weighing approximately 45 to 50 g at arrival were used. They were kept in separate cages in a room with a 12-hour light-dark cycle, with constant temperature (21 to 22OC) and humidity (50%). Procedure The rats (n = 63) were conditioned for a 6-day period before the study. During this period, the animals were fed a standard laboratory diet (R3, Ewos AB, Sijdertalje, Sweden) and water, both given ad libitum. This was followed by 36 hours of starvation, when only water was given. After starvation, the animals were refed for 24 hours with pellets and water, given ad libitum. Twenty-two rats were studied in the fed state, 22 after starvation, and 19 after refeeding. In addition, animals were observed for 48 and 72 hours of refeeding, with regard to body weight. The food intake of young rats shows very small interanimal variationI and was therefore not monitored. Body weight was determined each day. Protein synthesis and RNA content of muscle tissue were measured in each group. Before the muscle preparation, animals were anaesthetized at 9:00 AM by intraperitoneal injection of pentobarbital (Mebumal, 60 mg/mL, AC0 Liikemedel, Stockholm, Sweden) at a dose of 1 mL/kg body weight. Protein Synthesis in Intact Muscle and in Muscle Biopsy Specimens The intact extensor digitorum longus muscle (EDL) from one leg was carefully dissected, without stretching the muscle, and weighed. A biopsy was taken from the contralateral EDL muscle with a biopsy nipper. The incorporation of 14Gphenylalanine into protein was determined with the same principles as described by Fulks et al9 and Li et a14.‘0using 14-C-tyrosine. The procedure followed the identical method described by Svaninger et al.” The muscle preparations were incubated in McCoy’s cell culture medium containing 1.0 pmol/L of Hepes (N-2-hydroxyethylpiperazine-N’-2-ethansulfonic acid) and U-14-C-phenylalanine (lo6 dpm/3 mL) at +37’C. Phenylalanine was chosen, since it is not metabolized by skeletal muscle and equilibration is rapid between the intracellular pool and the medium.” The medium was adjusted to pH 7.4 and saturated with a 95% 0, and 5% CO, gas mixture before incubation. The incubation was terminated after 30, 60, and 90 minutes by adding ice-chilled 5% trichloroacetic acid (TCA). The muscle preparations were washed twice with the TCA to make sure that unincorporated label was removed. Lipids were extracted sequentially by adding chloroform:methanol 1: 1, ethanol:acetone 1:1, and finally pure ether. The muscle specimens were freeze-dried and the fat-free dry weight (fdw) was determined. The muscles were dissolved in a mixture of 0.5 mL distilled water and 1.5 mL Soluene 350 and radioactivity was determined in a scintillation counter. Protein synthesis was calculated as dpm/mg fdw per hour. The specific activity in the medium was used as an approximation for intracellular specific activity, as described elsewhere.” Since it has been speculated that the specific activity between the medium and the intracellular pool may vary with different physiological states, a separate study was performed in order to evaluate if the amino acid concentration in the medium could have influenced the results in the first study. Twelve rats were divided into three groups: fed, starved for 36 hours, and refed for 24 hours. Muscle biopsies were incubated in media containing one and 10 times the plasma concentration of all amino acids. The biopsies were treated as described above.
WAHREN.
AND EKMAN
Protein content was determined” in some muscles in order to quantify the correlation between protein content and fdw. The remaining part of the EDL muscle was immediately weighed and frozen in liquid nitrogen for RNA determination according to Munro and Fleck.19 Chemicals L-(U-14Qphenylalanine 0.55 mCi/mmol was obtained from New England Nuclear, Boston, MA. McCoy cell culture medium was from Flow Laboratories, London, UK. pH was held constant with Hepes, Sigma Chemical, St Louis, MO. The muscle tissue was dissolved in Soluene 350 from Packard Instrument, Maryden, CT. All chemicals used were of highest analytical purity. Data are presented as mean + SEM for each group of rats. Standard statistical methods were employed, using Student’s t test when applicable. When data were compared on relative basis, expressed as percent of their controls, the logarithm values were used and Wilcoxon unpaired test employed. RESULTS
Body Weight The rats gained approximately 5 k 1 g/d during the conditioning period prior to the study. After 36 hours starvation, the body weight had decreased by 28% or 22 + 1 g (Fig 1). Following 24 hours of refeeding, the animals had gained 15 + 1 g or 26%. After 2 more days, the rats had gained an additional 30 * 1 g or 42%. Protein Synthesis Incorporation of 14-C-phenylalanine into muscle protein was found to be a linear function of time for at least 90 minutes in both intact muscles and muscle biopsies. This was observed in all feeding conditions (Fig 2). However, the in-
0
2
4
6
6
10
12
Body weight (g) during conditioning period. Fig 1. hours of starvation, and after 3 days of refeeding.
Days
after
36
1115
PROTEIN SYNTHESIS IN SKELETAL MUSCLE
STARVED 36h
REFED 24h
-
‘“1
Fig 2. Incorporation and refed states.
of 14-C-phenylalanine
into protein fdpm/mg
corporation of 14-C-phenylalanine was consistently lower in the muscle biopsies compared with the intact muscle (Fig 2). Protein synthesis as evaluated by the incorporation of 14-C-phenylalanine in fed rats was 609 2 32 dpm/mg freeze-dried weight (fdw) per hour in intact muscles and 268 + 22 dpm/mg fdw per hour in muscle biopsies. After 36 hours of starvation the incorporation was significantly decreased in both preparations, 200 2 21 and 113 + 7 dpm/mg fdw per hour, respectively. After 24 hours of refeeding, protein synthesis was significantly increased compared with the fed and fasted states, both in intact muscles, 831 + 46 dpm/mg fdw per hour, and in muscle biopsies, 355 + 20 dpm/mg fdw per hour (Fig 2). Since the specific aim of this study was to compare the relative change in protein synthesis in intact muscles and muscle biopsies under different feeding conditions, the values were expressed as percent of those obtained in the fed state (Fig 3). Both muscle preparations showed a significant decrease to approximately 40% of the initial value, 33% * 10% in the intact muscles and 42% f 6% in the muscle biopsies after the starvation period. After 24 hours of refeeding, an overshoot to approximately 135% was seen in both preparations, 136% t 6% and 133% + 6%, respectively (Fig 4). Thus, the relative change in protein synthesis was similarly reflected in intact muscle and in the muscle biopsy material. In the second study, the relative change in protein synthesis were similar, when muscle biopsies were incubated in medium containing one and 10 times plasma amino acid concentrations. In the medium containing one time the plasma amino acid concentration the incorporation decreased to 50% after 36 hours of starvation and increased to 126% after 24 hours of refeeding. When using an incubation medium containing 10 times the plasma amino acid concentration, the incorporation was 57% and 113%, respectively. Thus, no statistical difference was seen between the two studies.
fdw) in intact muscle (ml and in muscle biopsy
1El)in the
fed, starved,
DISCUSSION It has been suggested that muscle protein synthesis measured in vitro in muscle biopsy material may be used to assess relative changes in the rate of protein synthesis during refeeding in cancer and postoperative states. This notion has not met with general acceptance.16 The reluctance to accept muscle biopsy material for measurements of protein synthesis is in contrast to the general use of the intact EDL muscle in rats for evaluating the influence of varying nutritional and hormonal stimuli on muscle protein synthesis.“,‘2.‘4.20 Therefore, the purpose of the present study was to compare, in the same animal, measurements of protein synthesis by the two methods. Starvation and refeeding were chosen as two conditions under which the two methods may be compared,
150$ 1 100
75
50
25
0i RNA Content The RNA content was significantly decreased, from 21.6 f 0.70 mg/g protein in the nonstarved rats to 14.5 + 0.41 mg/g protein, or 33%, after 36 hours of starvation. In contrast to the increased incorporation of 14-C-phenylalanine, the RNA content remained low, 14.8 f 0.48 mg/g protein, after 24 hours refeeding (Fig 4).
FED
STARVED
REFED
36 h
24 h
Fig 3. Protein synthesis after 36 hours of starvation and 24 hours of refeeding, relative to normal feeding in intact muscle (B) and in muscle biopsy @). Asterisks denote that the difference are caused by random factors, l*P < .DDDl if compared with the fed state; lP < .Ol if compared with the fed state (Wilcoxon unpaired test).
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MAGNUSSON,
25
I-
i-
l-r-
FED
STARVED 36 h
REFED 24 h
RNA content in muscles in rats, after 36 and after 24 of refeeding. Values are expressed as mg RNA/g the difference are caused by random factors, l*f -C .OOOl if compared with the fed state (Student’s unpaired t test). Fig 4.
since their respective effects on protein synthesis are well documented.*’ Rats weighing between 56 and 74 g were used, because 70 g is a minimal weight for survival during 24 hours of starvation and animals in this weight range provide EDL preparations that are small enough for optimal incubation conditions.22 A 30% decrease in body weight was observed during the starvation period, corresponding to the findings shown by Li et al.4 During the same period of starvation, they showed a 15% decrease in EDL wet weight. However, the dry weight to wet weight ratio did not change, indicating a loss of nonprotein components, such as glycogen, fat, and RNA. Since the metabolic rate is approximately 10 to 20 times higher in small rodents than in man,’ the 36-hour starvation period of the present study can be compared with long-term starvation in man. Therefore, the direction of the changes in protein synthesis shows a pattern similar to that in starved and refed man.* This study demonstrates that the muscle biopsies decreased their amino acid incorporation into protein by 58% during the starvation period and increased it by 33% during refeeding, compared with the values obtained in the fed state. The corresponding changes for the intact muscle were 67% decrease during starvation and 36% increase during refeeding. This demonstrates that the two procedures for evaluating protein synthesis in muscle are equally useful for assessing relative changes in amino acid incorporation during
WAHREN, AND EKMAN
starvation and refeeding. The metabolic intervention in the animals must be regarded as substantial, as reflected in the changes in body weight and incorporation capacity. It therefore remains to be determined whether the two methods are equally capable of assessing moderate to minor changes in protein synthetic capacity. This study also provides information regarding the relationship between protein synthesis and RNA content during starvation and refeeding. In both respects there was a significant decrease after 36 hours of starvation, but during the refeeding period the trends diverged, with an increase in protein synthesis and a persistently low RNA content. It has been shown that short-term refeeding results in an acute shift in the relative polyribosomal distribution.’ The primary response to refeeding seems to be a stimulation of the activity of RNA present in the cell; secondarily new synthesis of RNA takes place. While changes in RNA content appear adequately to reflect the depression of protein synthesis during starvation, no response to short-term refeeding was observed. It remains to be determined whether, during refeeding beyond 36 hours, changes in RNA content may reflect protein synthesis. Protein synthesis in the present study was expressed as dpm/mg fdw per hour. In some muscles protein content and fdw were compared. The fdw overestimated protein content by 10% i- 1% compared with protein determination. Since the difference was constant, protein synthesis expressed as dpm/mg fdw was considered to be as precise as dpm/mg protein. 14Gphenylalanine was chosen for the incorporation measurements, since it rapidly equilibrates between the medium and the intracellular pool and is neither synthesized nor degraded in the muscle tissue.’ However, neither intact muscle nor muscle biopsy preparations show the same specific activity in the two compartments, when using an incubation medium containing plasma concentrations of all amino acids.“js” If the muscle biopsy material is incubated in a medium containing 10 times the plasma concentrations of amino acids, the specific activity between the medium and the intracellular pool becomes equal.16 In the present study, we chose an amino acid solution with concentrations of amino acids similar to those of plasma. The reason for this was that an incubation medium with supraphysiological amino acid concentrations may in itself have an impact on the protein synthesis in that, for instance, leucine is considered to exert a stimulatory effect on protein synthesis.9.20 At physiological concentrations of amino acids in the medium, the ratio between the specific activities in the medium and the intracellular pool is similar in the intact muscle and the muscle biopsy material, respectively.‘6 Therefore, changes in incorporation rate in the two preparations cannot be explained by differences in the specific activity. It has previously been suggested that the specific activity between the medium and the intracellular pool will vary with physiological states. Therefore, comparison of muscle biopsies incubated in only one time the plasma amino acid concentration could be misleading, when different physiological states are compared. This was addressed by comparing the relative incorporation rate between biopsies incubated in
PROTEIN SYNTHESIS
IN SKELETAL MUSCLE
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and 10 times the plasma concentrations in fed rats and after starvation and refeeding. These results indicate that the observations made at physiological concentrations cannot be explained by differences in specific activity during various nutritional states. The amino acid incorporation capacity was found to be approximately 2.3 times greater in the intact muscle compared with the muscle biopsy material. This demonstrates the quantitative importance of maintaining the integrity of the muscle for protein synthesis evaluation. However, when investigating various nutritional, hormonal, or other metabolic interventions, it is the relative change in protein synthesis rather than the absolute values that is of imporone
tance, since the relative value describes the direction and magnitude of the changes in protein synthesis regardless of the absolute incorporation of labeled amino acids. In conclusion, the present results demonstrate that muscle biopsy specimens and intact muscle are equally useful in the estimation of changes in muscle protein synthesis in response to starvation and refeeding. Further work is required to define the ability of the two methods to detect small alterations in protein synthesis. The current findings validate the use of the biopsy procedure, thereby facilitating measurements of protein synthesis in human skeletal muscle under varying clinical conditions,
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