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Leucine oxidation in rat muscle, heart, and liver homogenates
BIOCHEMICAL
MEDICINE
Leucine
G. LYNN Department
15,
306-310
Oxidation
DOHM,
in Rat Muscle, Homogenates
WILLIAM
of Biochemistry.
(1976)
E. BROWN,
School of Medicine, North Carolina Received
April
and Liver
AND HISHAM
East 27834 7.
Heart,
Carolinu
A.
Uni\,ersity.
BARAKAT
tirern~~i//~~.
1976
Although it is now well established that skeletal muscle tissue can oxidize branched chain amino acids (l-3), the contribution of amino acid oxidation to the overall energy balance of muscle is not known. In muscle fibers suspended in a medium containing amino acids (no other substrates) at concentrations approximating intracellular levels, Beatty ef al. (4) found that amino acid oxidation accounted for about 20% of the CO, production. Adibi et al. (5) reported that leucine was oxidized at a more rapid rate than palmitate for muscle tissue slices that were incubated in a medium in which the concentration of leucine and palmitate were equal in terms of “carbon content.” Although these studies indicate that amino acid oxidation does take place at an appreciable rate relative to other energy-producing pathways, it is difficult to quantitate results from tissue slices and intact muscle fibers because the resultant CO, production can be affected by the rate of transport of the radioactive substrate into the cell and the dilution of label with nonlabeled substrated inside the cell. For this reason, we undertook the present study to compare the rate of L-[UJ4C]leucine oxidation in homogenates to previously determined rates of oxidation of fatty acids. In addition, the activity of leucine-cyketoglutarate transaminase and the oxidation rate of L-[ I-14C]leucine were determined to investigate the rate-limiting step in the oxidation of leucine, METHODS
Male Holtzman rats (Masidon, Wisconsin), weighing 300-400 g, were sacrificed by decapitation and their hearts, livers, and muscles (gastrocnemius and quadriceps) were excised and suspended in ice-cold homogenization medium (10 mM potassium phosphate, pH 7.5, containing 1 mM EDTA). The tissues were pressed through a tissue press, gently homogenized in a glass-glass Ten Broeck (A. H. Thomas. Philadelphia, Pa.) homogenizer and filtered through cheesecloth. Such a procedure was followed in order not to affect the activity of a-ketoisocaproic acid dehydrogenase (6). Boiled tissue homogenates were used for blanks. 306 Copyright All
rights
t2
1976
of reproduction
by
Academic m any
Press,
Inc.
form
reserved
LEUCINE
OXIDATION
IN HOMOGENATES
307
The assay for leucine oxidation was essentially the method of McFarlane and Von Holt (7) and Sketcher et al. (6). The incubation medium consisted of 0.2 mM thiamine pyrophosphate, 1 mM ATP, 0.1 mM malate, 0.1 mM CoA, 2 mM MgCl,, 1 mM dithiothreitol, 0.1 mM NAD+, 10 mM L-[ 1-14C]leucine or L-[U-14C]leucine (0.05 #Zi/~mole) and buffer (25 mM disodium hydrogen phosphate and 25 mM potassium dihydrogen phosphate at pH 7.5) in a final volume of 2.0 ml. The rate of leucine oxidation was measured by collecting r4C0, that was produced during the incubation period (60 min at 37°C) as previously described (8). For determination of transamination (6). fresh caps and center wells were placed on the incubation vessels that contained [lJ4C]leucine. Six milliliters of 4 N H,SO, saturated with CeSO, were injected into the reaction vessels and the reaction was incubated for 60 min at 37°C. The CO, produced by chemical decarboxylation of a-ketoisocaproic acid was trapped and counted. RESULTS AND DISCUSSION
One of the major objectives of this study was to investigate the relative importance of amino acids as an energy source in muscle. Previous investigators have attempted to elucidate this question by determining the rate of radioactive CO, production after incubating muscle fibers, muscle slices, or diaphragm with various ‘*C-labeled amino acids and/or other substrates. However, the rate of 14C0, production from these tissue preparations is dependent not only on the rate of oxidation of the substrate but also on the rate of transport of the substrate into the cell and the dilution of the isotope with unlabeled substrate of the tissue. To circumvent these difficulties we have determined the rate of oxidation of [‘4Clleucine in homogenates under assay conditions that give maximal rates of production of 14C0,. Since liver has been considered one of the major organs involved in amino acid oxidation, it was of interest to compare leucine oxidation rates in heart and skeletal muscle to that of liver. The rate of oxidation of L-[UJ4C]leucine was not greatly different for heart and liver. However, the oxidation rate in these two tissues was 40-50 times greater than in muscle. By using the maximal capacity for leucine oxidation (Table 1) and assuming that muscle constitutes 45%, liver 4%, and heart 0.5% of the total body weight, the total capacity of each tissue to oxidize leucine was calculated. The order of total capacity for leucine oxidation was liver, muscle, and heart. We previously reported (8) the oxidation rate of palmitate in muscle and heart homogenates under optimal conditions (5.5 ? 0.2 nmoles/min/g for muscle and 66.3 ? 3.7 nmoles/min/g for heart). Comparing the oxidation rates revealed that the oxidation of leucine (Table 1) was only 3.4 % that
308
DOHM. BROWN AND BARAKA’I TABLE
I
OXIDATION OF [I-WILEUCINE AND [U-14C]L~u( INE. AND LEUCINE-CX-KETOGLUTARATE TRANSAMINASE ACTIWTY OF MUSCLE, HEART, AND LIVER HOMOGENATES
Activity (nmolesiminig
Tissue Muscle Heart Liver
Transamination 79.5 2 22.1* 909 k 52 42.6 2 4.5
tissue)
[I-‘“C] leucine oxidation ____~..-.-..--0.456 + 0.135 44.7 ‘-c 5.7 30.1 I!I 6.4
Percentage 1I.,-‘“Cl / U-14C]leucine oxidation leucine oxidation irom C-I” __..--..-~~.- _.... --_.~~-~-.
0.192 _c 0.035 IO.? +_ 0.6 7.70 -t 0.51 ___~
40 13 bS _I_-_---.
n The amount of CO, from C-l of [U-‘4C]leucine oxidation was calculated by dividing the rate of CO, production from [ I-“Qleucine oxidation by 6. * Values are mean 2 SE for at least six observations.
of palmitate in muscle and 15% in heart. These observations suggest that under normal conditions leucine oxidation would supply only a small amount of the energy requirements of skeletal muscle, but a larger amount of energy in heart. Since isoleucine, valine, alanine, glutamate, and aspartate are also oxidized by muscle (9), the contribution of all amino acids may be larger. After starvation (5,9) and exercise training (Brown and Dohm, unpublished observation) the capacity for leucine oxidation in muscle is increased. Under these conditions, amino acid oxidation may make an appreciable contribution toward supplying the energy demands of the muscle. The activity of leucine-a-ketoglutarate transaminase and the oxidation rate of L-[1-14C]leucine were assayed to determine if either the transaminase or cy-ketoisocaproic acid dehydrogenase enzymes are rate limiting in the oxidation of leucine. The transaminase activity was approximately 175 times higher than the rate of oxidation of L-[1-14C]leucine in muscle and 20 times greater in heart. It thus appears unlikely that the transaminase is rate limiting in muscle or heart. In liver, however, the transaminase activity and the rate of oxidation of L-[I-i4C]leucine were very similar, suggesting that transamination was probably the ratelimiting step in leucine oxidation. These conclusions are in agreement with the preliminary report of Shinnick and Harper (10). The high activity of leucine-cr-ketoglutarate transaminase may be of significance to processes other than leucine oxidation. Felig and Wahren (11) found that alanine was released from the muscle in large quantities during exercise and they proposed the “glucose-alanine cycle.” Odessey
LEUCINE
OXIDATION
IN HOMOGENATES
309
et al. (12) and Ruderman and Berger (13) have found that alanine release from the muscle is increased when leucine is provided in the medium bathing the muscle. From their data Odessey et al. (12) suggest the existence of a branched chain amino acid-alanine cycle in muscle. Thus, the high activity of the transaminase is possibly significant in terms of providing an amino group for the transamination of pyruvate formed as a result of glycolysis. Several investigators (2,3) have compared the rates of oxidation of ~-[l-l~C]- and L-[U-14Clleucine to establish if the molecule is completely degraded and if a-ketoisocaproic acid dehydrogenase is the enzyme limiting the oxidation of leucine. We have calculated the percentage of the total COz evolved from [U-14C]leucine that is the result of the oxidation of the number one carbon atom (see Table 1). If no oxidation occurs after the a-ketoisocaproic acid dehydrogenase step, the value would be 100%. On the other hand, if cr-ketoisocaproic acid dehydrogenase is the rate-limiting step and the oxidation of the remainder of the molecule is rapid and complete, the value would theoretically be 17%. As pointed out by Odessey and Goldberg (2), this ideal value (17%) may not be obtained since degradation of [UJ4C]leucine produces many radioactive intermediates (acetate, acetoacetate, etc.) that will be diluted by a large amount of nonradioactive material. There was appreciable oxidation (60 and 27% in muscle and heart, respectively) beyond the a-ketoisocaproic acid dehydrogenase step and it seems possible that a-ketoisocaproic acid dehydrogenase may be the rate-limiting enzyme in leucine oxidation in muscle and heart. Since the values presented in Table 1 were obtained under assay conditions that give the maximum oxidation rate, it is difficult to draw conclusions about the rates of oxidation in the intact animal. The in vivo rate of leucine oxidation will depend on concentrations of substrates, cofactors, activators, etc. However, from these data it is clear that the maximum rate of oxidation in muscle is small for leucine compared to palmitate. In addition, the total capacity to oxidize leucine is greater in liver than in muscle despite the larger quantity of muscle. SUMMARY
This study was undertaken to investigate the rate of amino acid oxidation in homogenates of muscle, heart, and liver, and to investigate the rate-limiting enzyme in the oxidation of leucine. The rate of oxidation of leucine in homogenates was 3% the rate of palmitate oxidation for muscle and 15% for heart. The total capacity for leucine oxidation was largest in liver, followed by muscle, and then heart. Leucine-a-ketoglutarate transaminase appears to be rate limiting in liver but not muscle and heart.
310
DOHM.
BROWN AND BARAKAT
There was appreciable oxidation of leucine (2S-60%’ of total CO, produced) beyond the I-u-ketoisocaproic acid dehydrogenase reaction in muscle, heart, and liver. ACKNOWLEDGMENTS This research was supported in part by a cooperative research agreement (No. 11-i+ 1001-429) with the Agricultural Research Service, USDA. Beltsville, Md. The authors thank Ms. N. Leggett and Mr. C. Smith for technical assistance. and Ms. E. Jones for typing the manuscript.
REFERENCES 1. 2. 3. 4.
Manchester, K. L.. Biochim. Biophys. Actu 100, 295 (1965). Odessey, R., and Goldberg, A. L.. Amer. J. Ph~siol. 223, 1376 (1972). Buse. M. G.. Jursinic, S., and Reid. S. S., Biochrm. J. 148, 363 (1975). Beatty, C. H., Curtis, S.. Young, M. K., and Bocek, R. M.. Amer. J. Physioi.
227. 26X
(1974).
5. Adibi, S. A., Krzysik. B. A., Morse, E. L., Amin, P. N.. and Allen. E. R.. J. Lab. C71rr. Med.
83, 548 (1974).
6. Sketcher, R. D., Fern, E. B.. and James, W. P. T.. Bri/. .I. /Vu/r.. 31, 333 11974). 7. McFarlane. I. G., and Von Holt, C.. Biochem. J. 111, 565 t 1969). 8. Dohm, G. L., Huston, R. L., Askew. E. W., and Weiser. P. C.. ,tirttrr-. ./. I’h~.riol. 223, 783 (1972).
Goldberg, A. L., and Odessey. R.. Amer. J. Phy.rk~/. 223, 1384 t 1972). 10. Shinnick, F. L., and Harper. A. E.. Fed. Proc. 34. 880 (1975). 11. Felig, P., and Wahren. J., in “Muscle Metabolism during Exercise” (B. Pernow and B. Saltin, Eds.), p. 205. Plenum Press, New York. 1971. 12. Odessey, R., Khairallah. E. A., and Goldberg, A. L., J. Biol. C/I~IW. 249. 7623 t 1974). 13. Ruderman, N. B., and Berger. M.. J, Biol. Chem. 249, 5500 (1974).
9.
MEDICINE
Leucine
G. LYNN Department
15,
306-310
Oxidation
DOHM,
in Rat Muscle, Homogenates
WILLIAM
of Biochemistry.
(1976)
E. BROWN,
School of Medicine, North Carolina Received
April
and Liver
AND HISHAM
East 27834 7.
Heart,
Carolinu
A.
Uni\,ersity.
BARAKAT
tirern~~i//~~.
1976
Although it is now well established that skeletal muscle tissue can oxidize branched chain amino acids (l-3), the contribution of amino acid oxidation to the overall energy balance of muscle is not known. In muscle fibers suspended in a medium containing amino acids (no other substrates) at concentrations approximating intracellular levels, Beatty ef al. (4) found that amino acid oxidation accounted for about 20% of the CO, production. Adibi et al. (5) reported that leucine was oxidized at a more rapid rate than palmitate for muscle tissue slices that were incubated in a medium in which the concentration of leucine and palmitate were equal in terms of “carbon content.” Although these studies indicate that amino acid oxidation does take place at an appreciable rate relative to other energy-producing pathways, it is difficult to quantitate results from tissue slices and intact muscle fibers because the resultant CO, production can be affected by the rate of transport of the radioactive substrate into the cell and the dilution of label with nonlabeled substrated inside the cell. For this reason, we undertook the present study to compare the rate of L-[UJ4C]leucine oxidation in homogenates to previously determined rates of oxidation of fatty acids. In addition, the activity of leucine-cyketoglutarate transaminase and the oxidation rate of L-[ I-14C]leucine were determined to investigate the rate-limiting step in the oxidation of leucine, METHODS
Male Holtzman rats (Masidon, Wisconsin), weighing 300-400 g, were sacrificed by decapitation and their hearts, livers, and muscles (gastrocnemius and quadriceps) were excised and suspended in ice-cold homogenization medium (10 mM potassium phosphate, pH 7.5, containing 1 mM EDTA). The tissues were pressed through a tissue press, gently homogenized in a glass-glass Ten Broeck (A. H. Thomas. Philadelphia, Pa.) homogenizer and filtered through cheesecloth. Such a procedure was followed in order not to affect the activity of a-ketoisocaproic acid dehydrogenase (6). Boiled tissue homogenates were used for blanks. 306 Copyright All
rights
t2
1976
of reproduction
by
Academic m any
Press,
Inc.
form
reserved
LEUCINE
OXIDATION
IN HOMOGENATES
307
The assay for leucine oxidation was essentially the method of McFarlane and Von Holt (7) and Sketcher et al. (6). The incubation medium consisted of 0.2 mM thiamine pyrophosphate, 1 mM ATP, 0.1 mM malate, 0.1 mM CoA, 2 mM MgCl,, 1 mM dithiothreitol, 0.1 mM NAD+, 10 mM L-[ 1-14C]leucine or L-[U-14C]leucine (0.05 #Zi/~mole) and buffer (25 mM disodium hydrogen phosphate and 25 mM potassium dihydrogen phosphate at pH 7.5) in a final volume of 2.0 ml. The rate of leucine oxidation was measured by collecting r4C0, that was produced during the incubation period (60 min at 37°C) as previously described (8). For determination of transamination (6). fresh caps and center wells were placed on the incubation vessels that contained [lJ4C]leucine. Six milliliters of 4 N H,SO, saturated with CeSO, were injected into the reaction vessels and the reaction was incubated for 60 min at 37°C. The CO, produced by chemical decarboxylation of a-ketoisocaproic acid was trapped and counted. RESULTS AND DISCUSSION
One of the major objectives of this study was to investigate the relative importance of amino acids as an energy source in muscle. Previous investigators have attempted to elucidate this question by determining the rate of radioactive CO, production after incubating muscle fibers, muscle slices, or diaphragm with various ‘*C-labeled amino acids and/or other substrates. However, the rate of 14C0, production from these tissue preparations is dependent not only on the rate of oxidation of the substrate but also on the rate of transport of the substrate into the cell and the dilution of the isotope with unlabeled substrate of the tissue. To circumvent these difficulties we have determined the rate of oxidation of [‘4Clleucine in homogenates under assay conditions that give maximal rates of production of 14C0,. Since liver has been considered one of the major organs involved in amino acid oxidation, it was of interest to compare leucine oxidation rates in heart and skeletal muscle to that of liver. The rate of oxidation of L-[UJ4C]leucine was not greatly different for heart and liver. However, the oxidation rate in these two tissues was 40-50 times greater than in muscle. By using the maximal capacity for leucine oxidation (Table 1) and assuming that muscle constitutes 45%, liver 4%, and heart 0.5% of the total body weight, the total capacity of each tissue to oxidize leucine was calculated. The order of total capacity for leucine oxidation was liver, muscle, and heart. We previously reported (8) the oxidation rate of palmitate in muscle and heart homogenates under optimal conditions (5.5 ? 0.2 nmoles/min/g for muscle and 66.3 ? 3.7 nmoles/min/g for heart). Comparing the oxidation rates revealed that the oxidation of leucine (Table 1) was only 3.4 % that
308
DOHM. BROWN AND BARAKA’I TABLE
I
OXIDATION OF [I-WILEUCINE AND [U-14C]L~u( INE. AND LEUCINE-CX-KETOGLUTARATE TRANSAMINASE ACTIWTY OF MUSCLE, HEART, AND LIVER HOMOGENATES
Activity (nmolesiminig
Tissue Muscle Heart Liver
Transamination 79.5 2 22.1* 909 k 52 42.6 2 4.5
tissue)
[I-‘“C] leucine oxidation ____~..-.-..--0.456 + 0.135 44.7 ‘-c 5.7 30.1 I!I 6.4
Percentage 1I.,-‘“Cl / U-14C]leucine oxidation leucine oxidation irom C-I” __..--..-~~.- _.... --_.~~-~-.
0.192 _c 0.035 IO.? +_ 0.6 7.70 -t 0.51 ___~
40 13 bS _I_-_---.
n The amount of CO, from C-l of [U-‘4C]leucine oxidation was calculated by dividing the rate of CO, production from [ I-“Qleucine oxidation by 6. * Values are mean 2 SE for at least six observations.
of palmitate in muscle and 15% in heart. These observations suggest that under normal conditions leucine oxidation would supply only a small amount of the energy requirements of skeletal muscle, but a larger amount of energy in heart. Since isoleucine, valine, alanine, glutamate, and aspartate are also oxidized by muscle (9), the contribution of all amino acids may be larger. After starvation (5,9) and exercise training (Brown and Dohm, unpublished observation) the capacity for leucine oxidation in muscle is increased. Under these conditions, amino acid oxidation may make an appreciable contribution toward supplying the energy demands of the muscle. The activity of leucine-a-ketoglutarate transaminase and the oxidation rate of L-[1-14C]leucine were assayed to determine if either the transaminase or cy-ketoisocaproic acid dehydrogenase enzymes are rate limiting in the oxidation of leucine. The transaminase activity was approximately 175 times higher than the rate of oxidation of L-[1-14C]leucine in muscle and 20 times greater in heart. It thus appears unlikely that the transaminase is rate limiting in muscle or heart. In liver, however, the transaminase activity and the rate of oxidation of L-[I-i4C]leucine were very similar, suggesting that transamination was probably the ratelimiting step in leucine oxidation. These conclusions are in agreement with the preliminary report of Shinnick and Harper (10). The high activity of leucine-cr-ketoglutarate transaminase may be of significance to processes other than leucine oxidation. Felig and Wahren (11) found that alanine was released from the muscle in large quantities during exercise and they proposed the “glucose-alanine cycle.” Odessey
LEUCINE
OXIDATION
IN HOMOGENATES
309
et al. (12) and Ruderman and Berger (13) have found that alanine release from the muscle is increased when leucine is provided in the medium bathing the muscle. From their data Odessey et al. (12) suggest the existence of a branched chain amino acid-alanine cycle in muscle. Thus, the high activity of the transaminase is possibly significant in terms of providing an amino group for the transamination of pyruvate formed as a result of glycolysis. Several investigators (2,3) have compared the rates of oxidation of ~-[l-l~C]- and L-[U-14Clleucine to establish if the molecule is completely degraded and if a-ketoisocaproic acid dehydrogenase is the enzyme limiting the oxidation of leucine. We have calculated the percentage of the total COz evolved from [U-14C]leucine that is the result of the oxidation of the number one carbon atom (see Table 1). If no oxidation occurs after the a-ketoisocaproic acid dehydrogenase step, the value would be 100%. On the other hand, if cr-ketoisocaproic acid dehydrogenase is the rate-limiting step and the oxidation of the remainder of the molecule is rapid and complete, the value would theoretically be 17%. As pointed out by Odessey and Goldberg (2), this ideal value (17%) may not be obtained since degradation of [UJ4C]leucine produces many radioactive intermediates (acetate, acetoacetate, etc.) that will be diluted by a large amount of nonradioactive material. There was appreciable oxidation (60 and 27% in muscle and heart, respectively) beyond the a-ketoisocaproic acid dehydrogenase step and it seems possible that a-ketoisocaproic acid dehydrogenase may be the rate-limiting enzyme in leucine oxidation in muscle and heart. Since the values presented in Table 1 were obtained under assay conditions that give the maximum oxidation rate, it is difficult to draw conclusions about the rates of oxidation in the intact animal. The in vivo rate of leucine oxidation will depend on concentrations of substrates, cofactors, activators, etc. However, from these data it is clear that the maximum rate of oxidation in muscle is small for leucine compared to palmitate. In addition, the total capacity to oxidize leucine is greater in liver than in muscle despite the larger quantity of muscle. SUMMARY
This study was undertaken to investigate the rate of amino acid oxidation in homogenates of muscle, heart, and liver, and to investigate the rate-limiting enzyme in the oxidation of leucine. The rate of oxidation of leucine in homogenates was 3% the rate of palmitate oxidation for muscle and 15% for heart. The total capacity for leucine oxidation was largest in liver, followed by muscle, and then heart. Leucine-a-ketoglutarate transaminase appears to be rate limiting in liver but not muscle and heart.
310
DOHM.
BROWN AND BARAKAT
There was appreciable oxidation of leucine (2S-60%’ of total CO, produced) beyond the I-u-ketoisocaproic acid dehydrogenase reaction in muscle, heart, and liver. ACKNOWLEDGMENTS This research was supported in part by a cooperative research agreement (No. 11-i+ 1001-429) with the Agricultural Research Service, USDA. Beltsville, Md. The authors thank Ms. N. Leggett and Mr. C. Smith for technical assistance. and Ms. E. Jones for typing the manuscript.
REFERENCES 1. 2. 3. 4.
Manchester, K. L.. Biochim. Biophys. Actu 100, 295 (1965). Odessey, R., and Goldberg, A. L.. Amer. J. Ph~siol. 223, 1376 (1972). Buse. M. G.. Jursinic, S., and Reid. S. S., Biochrm. J. 148, 363 (1975). Beatty, C. H., Curtis, S.. Young, M. K., and Bocek, R. M.. Amer. J. Physioi.
227. 26X
(1974).
5. Adibi, S. A., Krzysik. B. A., Morse, E. L., Amin, P. N.. and Allen. E. R.. J. Lab. C71rr. Med.
83, 548 (1974).
6. Sketcher, R. D., Fern, E. B.. and James, W. P. T.. Bri/. .I. /Vu/r.. 31, 333 11974). 7. McFarlane. I. G., and Von Holt, C.. Biochem. J. 111, 565 t 1969). 8. Dohm, G. L., Huston, R. L., Askew. E. W., and Weiser. P. C.. ,tirttrr-. ./. I’h~.riol. 223, 783 (1972).
Goldberg, A. L., and Odessey. R.. Amer. J. Phy.rk~/. 223, 1384 t 1972). 10. Shinnick, F. L., and Harper. A. E.. Fed. Proc. 34. 880 (1975). 11. Felig, P., and Wahren. J., in “Muscle Metabolism during Exercise” (B. Pernow and B. Saltin, Eds.), p. 205. Plenum Press, New York. 1971. 12. Odessey, R., Khairallah. E. A., and Goldberg, A. L., J. Biol. C/I~IW. 249. 7623 t 1974). 13. Ruderman, N. B., and Berger. M.. J, Biol. Chem. 249, 5500 (1974).
9.