BIOCHEMICAL
MEDICINE
20, 395-403
(I9781
Pyruvate Oxidation in Rat and Human Skeletal Muscle Mitochondria
A.J.
H. BOOKELMAN,* J. M. F. TRIJBELS,* R. C. A. SENGERS,* M. JANSSEN,* J. H. VEERKAMP,t AND A. M. STADHOLJDERS~ *Department of Pediutrics, tDepurtment of Biochemistr!. und SDepurtment of Submicroscopic Morphology. University qf Nijmegen. Nijmegen, The Netherlands Received
March
15, 1978
A vast amount of knowledge has accumulated during the past years regarding pyruvate oxidation. Regulation of the isolated enzyme as well as regulation of pyruvate oxidation in intact mitochondria have been s&died extensively (l-7). The majority of these studies has been performed with liver and heart muscle mitochondria. There have been hardly any studies concerning pyruvate oxidation in skeletal muscle mitochondria. The investigations performed so far are studies about isolation procedures and hence are only concerned with oxygen uptake and respiratory control (8-13). Pyruvate oxidation in mitochondria isolated from various tissues and species is known to be stimulated by citric acid cycle intermediates and carnitine (4,14-18). In this paper the stimulatory effects of citric acid cycle intermediates and carnitine on pyruvate oxidation in skeletal muscle mitochondria were studied in order to obtain information about the ratelimiting step during [I-r4C]pyruvate oxidation. The results obtained with rat skeletal muscle mitochondria are compared with those obtained with human skeletal muscle mitochondria, and their application for the diagnosis of a defect in human skeletal muscle pyruvate oxidation is discussed. MATERIALS AND METHODS Preparation qf skeletal muscle mitochondria. Rat skeletal muscle mitochondria were prepared from the hind leg of male Wistar rats, weighing 150-200 g and fasted overnight. Human skeletal muscle was obtained from patients with no known neuromuscular disease. The muscle specimens 395
0006-294417810203-0395$02.00/O CopyrIght @ 1978 by Academic Pres. Inc. All nghtc of reproduction in any form reserved
396
BOOKELMAN
ET AL.
were placed in ice-cold isolation medium containing 250 mM sucrose, 2 mM EDTA, 1OmM Tris, 50 IU of heparin/ml, final pH 7.4 (SETHmedium). All further operations were carried out at O-4”. Muscle tissue was freed from fat and connective tissue, cut into very fine pieces with scissors, and suspended in SETH-medium (1 g/10 ml). Homogenization was performed by hand with a Teflon-glass Potter-Elvehjem-type homogenizer. The resulting homogenate was centrifuged for 10 min at 6OOg to remove nuclei and cell debris. After repeating this centrifugation, mitochondria were isolated from the resulting supernatant by centrifugation for 10 min at 14,000g. The mitochondrial pellet was rinsed with SET-medium (which is SETH medium from which heparin is omitted) to remove the fluffy layer, resuspended in SET-medium, and centrifuged again for 10 min at 14,OOOg. The mitochondria were finally taken up in SET-medium. Oxygen consumption of the mitochondria, measured as described by Max et al. (13), showed respiratory control indices higher than 5, and P/O ratios between 2.8 and 3.0 with pyruvate and malate as substrates, indicating tight coupling between oxidation and phosphorybtion. Incubations and ussa~~. Mitochondria were incubated in small sealed glass vials at 37” in a shaking water bath. The basic incubation medium contained in a final volume of 500 ~1: 30 mM potassium phosphate, 2 mM EDTA. 10 mM Tris. 5 mM MgCl,, 75 mM KCI, 25 mrvt sucrose, 20 mrvr glucose, 2 mM ADP, and 1-2 units of hexokinase. The final pH was 7.4. Concentrations of substrates are given in the legends to figures and tables. Incubations were started by addition of 20-30 ,ug of mitochondrial protein. Small plastic tubes containing 200 ~1 of Hyamine were inserted into the vials in order to trap ‘*CO, produced during oxidation of [l-‘*Cl pyruvate (19). Incubations were terminated, after 10 or 15 min, by addition of 200 ~1 of 3 M HCIO,. The vials were left for an additional 30 min in the shaking water bath to ensure complete trapping of the 14C0, by the Hyamine. Thereafter, the small plastic tubes with the Hyamine were transferred into 20-ml glass counting vials containing 10 ml of toluene and 40 mg of Omnifluor. Radioactivity was measured in a Nuclear Chicago Mark 1 liquid scintillation counter. Under all conditions tested, ‘4c0, production from [I-Wlpyruvate was linear with respect to time and the amount of protein present. Pyruvate was determined enzymatically according to Czok and Lamprecht (20). Protein was determined by the method of Lowry et al. (21) using bovine serum albumin as a standard. Statistical analyses were performed with Student’s t test. Chemicals. The sodium salt of [ 1-‘*C]pyruvate was purchased from the Radiochemical Centre, Amersham, England, and stored freeze-dried under nitrogen in sealed ampoules at -20” to minimize decomposition (22). Further reagents were purchased as follows: L(--)-carnitine and
PYRUVATE
OXIDATION
397
IN MUSCLE
hexokinase (freeze-dried, 40 units/mg) from Koch Light, Colnbrook. gland: rotenone and ADP from Boehringer und Sohne, Mannheim, Germany; Omnifluor from NEN chemicals GmbH, Frankfurt, West many; and the hydroxide of Hyamine 10X from Packard Instrument Chicago, Ill., U.S.A. All other chemicals were of the purest grade mercially available.
EnWest GerCo.. com-
RESULTS Stimulation of pyruvate oxidation by citric acid cycle intermediates. The effect of increasing concentrations of malate, fumarate, and succinate on the rate of pyruvate oxidation by rat skeletal muscle mitochondria is shown in Fig. 1. All the citric acid cycle intermediates tested stimulate pyruvate oxidation, although the effect of fumarate and succinate is smaller than that observed with malate. In all instances a final concentration of 1 mM of the added citric acid cycle intermediates is enough to approximate the maximal stimulatory effect which can be obtained with a particular intermediate. In the presence of malate the rate of pyruvate oxidation measured by the production of 14C0, from [l-14C]pyruvate equals the rate of pyruvate
-,-
”
0.5 cltrlc
acid
1.0 cycle
2.0 intermedIate
[mM]
FIG. 1. Stimulation of pyruvate oxidation by citric acid cycle intermediates. Rat skeletal muscle mitochondria (54 cog of protein/ml) were incubated in the basic incubation medium supplemented with I mM pyruvate (0.15 &i of [I-‘4Clpyruvate). Malate, fumarate, and succinate were present in final concentrations shown in the figure. 0-O. malate: X-X. fumarate: O-0, succinate.
398
BOOKELMAN
ET AL.
disappearance from the incubation medium measured enzymatically, indicating that all the pyruvate taken up by the mitochondria is oxidized to CO, (Table 1). Stirnulution of pynrvcrte oxidation by cnrnitine. The rate of 14C0, production from [l-‘“Clpyruvate is not only increased by addition of citric acid cycle intermediates but also by addition of carnitine to the mitochondria. Acetyl-CoA formed by the pyruvate dehydrogenase complex can be transferred to carnitine by the action of carnitine acetyltransferase (23,241 to form acetylcarnitine and to regenerate mitochondrial CoASH, thereby relieving inhibition of the pyruvate dehydrogenase complex by acetylCoA (25). Carnitine acetyltransferase activity has been shown to be present in muscle mitochondria (26). The effect of carnitine on pyruvate oxidation can be seen in Fig. 2. The double reciprocal plot of oxidation rate versus carnitine concentration shows biphasic kinetics. Two apparent K,, values of 0.2 m and 3.7 mM can be calculated corresponding with a V max of 4.1 and 16.1 pmoles/mg of protein .hr, respectively. The V,,, reached at infinite carnitine concentration approximates closely the oxidation rate obtained in the presence of I mM malate which was 17.0 pmoles/mg of protein.hr in this experiment. In the presence of malate no acetate formation was observed. The main products formed in the presence of carnitine were acetylcarnitine and acetate, the latter probably resulting from hydrolysis of acetylcarnitine. Pynrvcrte oxidcltiotl ,cithout addition of citric acid cycle intermediates or camitine. An attempt was made to investigate whether the low rate of pyruvate oxidation measured when no citric acid cycle intermediates or carnitine were added was due to pyruvate oxidation supported by endogenous citric acid cycle intermediates or due to some nonspecific decarboxylation of the [ I-%]pyruvate. Therefore, the effect of arsenite, an inhibitor of the pyruvate dehydrogenase complex (27) was studied. Figure 3 shows the effect of arsenite on pyruvate oxidation measured without TABLE RATE
Parameter Pyruvate Pyruvate
OF PYRUVATE
UPTAKE
I AND
Rate (pmoles/mg uptake oxidation
OXIDATION”
of protein
.hr)
16.3 t 1.9 (4) 15.8 2 1.2 (6)
I’ Rat skeletal muscle mitochondria were incubated in the basic incubation medium supplemented with 1 mM pyruvate (0.15 &i of [l-W] pyruvate) and 1 mM malate. Pyruvate uptake was measured as the difference in pyruvate concentration in the neutralized deproteinized perchloric acid extracts of the medium before and after incubation. Pyruvate oxidation was measured as described in Materials and Methods. Values shown are means ? SD of the number of experiments given in parentheses.
PYRUVATE
OXIDATION
399
IN MUSCLE
0
i
i 1
[carnltlne] FIG. 2. Lineweaver-But-k skeletal muscle mitochondria. basic incubation medium.
4 mM-’
plot for carnitine stimulation of pyruvate oxidation in rat Pyruvate ( ImM) (0. I5 &i of [I-‘4Clpyruvate) was added to the
additions, with carnitine and with malate. A prompt inhibition of pyruvate oxidation by low concentrations of arsenite is observed in the presence of malate or carnitine. However, virtually no inhibition is observed when pyruvate oxidation is measured without addition of citric acid cycle intermediate or carnitine. Moreover, the residual activity measured under all three conditions is the same. This indicates that part of the pyruvate
161
0
01 arsenate
0.2 [ mM I
FIG. 3. Inhibition of pyruvate oxidation by arsenite. Rat skeletal muscle mitochondria (115 fig of protein/ml) were incubated in the basic incubation medium supplemented with 1 mM pyruvate (0.15 &i of [I-‘4Clpyruvate). Further additions were: O-0, none: O-O, 5 mM Carnitine; X-X, 1 mM malate.
400
BOOKELMAN
ET AL.
TABLE EFFECT
OF INHIBITORY CITRIC
ON PYRUVATE ACID
CYCLE
2
OXIDATION
IN THE
INTERMEDIATES
ABSENCE
OF ADDED
OR CARNITINE”
Addition
Pyruvate oxidation rate (pmoles/mg of protein. hr)
None Rotenone Malonate Arsenite
1.5 0.2 0.5 1.1
” Rat skeletal muscle mitochondria (46 &ml) were incubated in the basic incubation medium supplemented with 1 mM pyruvate (0.15 &i of [I-‘Qpyruvate). Incubations were terminated after 15 min. Inhibitor concentrations were I-&ml rotenon, 5 mM malonate, 0.2 mM arsenite.
oxidation measured is insensitive to arsenite inhibition and hence this activity may not represent pyruvate dehydrogenase activity. This conclusion is not supported by the effects observed with rotenone or malonate as shown in Table 2. The residual activity is completely destroyed by heating of the mitochondria (3 min at 100’) prior to incubation. Pyruvate oxidation in rat and human skeletal muscle mitochondria. In Table 3 the stimulatory effects on pyruvate oxidation of the citric acid TABLE EFFECT
OF CITRIC
ACID
PYRUVATE
CYCLE
3
INTERMEDIATES
OXIDATION
IN RAT
MUSCLE
AND AND
CARNITINE
HUMAN
ON THE
RATE
OF
SKELETAL
MITOCHONDRIA”
Pyruvate oxidation rate
Addition
Rat (pmoles/mg of protein .hr)
Human (~moles/mg of protein .hr)
None Malate Fumarate Succinate Carnitine Carnitine + malate Carnitine + fumarate Carnitine + succinate
1.1 15.8 7.5 10.7 8.3 16.4 13.0 14.7
2.0 7.5 3.0 4.9 7.9 8.8 7.1 7.3
2 T 2 2 2 2 f 2
0.2 (5) 1.2 (6) 1.1 (4) 0.6 (4) 0.8*(5) 1.7 (5) 1.9*(4) 1.4*(4)
of 0.8 (5) + 3.5 (6) 2 1.3 (4) * 2.0 (4) k 3.8*(6) 2 4.3 (5) z!r 3.3 (4) 2 3.8 (4)
Relative (%) 25? 100 45 It 732 105 * IlOk 106 ” 107 t
3 10 5 9* 7 12* 9*
4 Mitochondria were incubated in the basic incubation medium supplemented with I mM pyruvate (0.15 &i of [l-‘Flpyruvate). Additions of citric acid cycle intermediates (1 mM) or carnitine (5 mM) were made as indicated. Values shown are means * SD of the number of experiments given in the parentheses. The significance of the difference in oxidation rate in the presence and absence of carnitine was calculated. * P < 0.05.
PYRUVATE
OXIDATION
IN MUSCLE
401
cycle intermediates malate, fumarate, and succinate are compared with the effect of carnitine in rat and human skeletal muscle mitochondria. The values for the rate of pyruvate oxidation obtained with human skeletal muscle mitochondria are presented in two manners. Both the absolute values (pmoles/mg of protein *hr) and the relative values (expressed as percentage of those obtained with 1 mrvr malate) are presented. In the latter manner standard deviations were greatly reduced. Both in rat and human skeletal muscle mitochondria maximal stimulation of pyruvate oxidation is achieved by malate although this effect is greater in rat than in human skeletal muscle mitochondria. A decrease in stimulatory effect in the order malate> succinate> fumarate is observed both with rat and human muscle mitochondria. Addition of carnitine results in a stimulation of pyruvate oxidation rate which is in rat skeletal muscle mitochondria half of that observed with malate while in human skeletal muscle mitochondria the same stimulation is achieved as with malate. In skeletal muscle mitochondria of both species pyruvate oxidation measured in the presence of fumarate or succinate can be further stimulated by addition of carnitine. In the presence of malate no further stimulation by carnitine is observed. DISCUSSION Pyruvate oxidation in skeletal muscle mitochondria incubated with I mrvf pyruvate and an excess of ADP but without addition of a citric acid cycle intermediate or carnitine is limited by the availability of oxaloacetate. This can be concluded from the stimulatory effect of citric acid cycle intermediates and carnitine. The differences which are observed between the stimulatory effects of malate, fumarate, and succinate (Fig. 1) are probably caused by differences in the permeability of the mitochondrial membrane toward these intermediates. The role of acceptor for acetyl-CoA generated by the action of the pyruvate dehydrogenase complex is not restricted to oxaloacetate but can also be fullfilled by carnitine (14, 16, 18, 23, 24). Ths biphasic kinetics of the stimulation of pyruvate oxidation by carnitine shown in Fig. 2 points to the action of two different enzymes. The V,,, reached at infinite carnitine concentration closely approximates the activity found in the presence of malate. So, both malate and carnitine are able to stimulate pyruvate oxidation to the same extent although the effect of malate is reached at far lower concentrations. Pyruvate oxidation measured without addition of citric acid cycle intermediate or carnitine is supported by endogenous citric acid cycle intermediates. This can be concluded from the observed inhibition by malonate (Table 2). Spontaneous decarboxylation cannot be responsible for this residual activity since the latter is inhibited by malonate,
402
BOOKELMAN
ET AL.
rotenone. and heating of the mitochondria. The partial insensitivity toward arsenite was attributed in a recent report concerning kidney mitochondria (28) to an overestimation of the amount of evolved 14C0,. However, since arsenite is a water-soluble reagent and the pyruvate dehydrogenase complex is bound to the inner mitochondrial membrane, partial inaccessibility of the pyruvate dehydrogenase complex toward arsenite in intact mitochondria is an alternative explanation. Citric acid cycle intermediates and carnitine stimulate pyruvate oxidation both in rat and human skeletal muscle mitochondria. Large standard deviations were obtained with human skeletal muscle mitochondria when the activities are expressed on protein base (Table 3). This was reported previously (29). Therefore, the problem of finding a better reference base to express the activities found in human material needs further investigation. The stimulatory effect of carnitine on pyruvate oxidation in the presence of fumarate or succinate indicates that with these two citric acid cycle intermediates the availability of the acetyl-CoA acceptor, oxaloacetate, is still rate-limiting for maximal pyruvate dehydrogenase activity. Since no further stimulation of pyruvate oxidation can be achieved by carnitine in the presence of malate, the rate of acetyl-CoA production is the rate-limiting step during [I-‘4C]pyruvate oxidation under this condition. The stimulation of pyruvate oxidation by carnitine is equal to that observed with malate in human skeletal muscle mitochondria in contrast to that in rat skeletal muscle mitochondria. This suggests that carnitine acetyltransferase activity is relatively greater in human skeletal muscle mitochondria. Parallel incubations of human skeletal muscle mitochondria with [ I-‘“Clpyruvate plus malate or carnitine will be the best means of obtaining information about the capacity of human skeletal muscle mitochondria to oxidize pyruvate. It not only permits the diagnosis of an impaired rate of pyruvate oxidation, but it also discriminates between a defect in pyruvate dehydrogenase and an impaired capacity of the mitochondria to oxidize acetyl-CoA via the citric acid cycle. In case of a defect in the respiratory chain, it can be expected that pyruvate oxidation will be impaired both with malate and with carnitine added. Then further investigations are indicated to determine whether the defect in pyruvate oxidation is due to a defect in pyruvate dehydrogenase or whether the defect is secondary to a defect in the respiratory chain. SUMMARY
Pyruvate oxidation in rat and human skeletal muscle mitochondria was studied by measuring the rate of ‘CO, production from [lJ4C]pyruvate in the presence of 1 mM pyruvate, an excess of ADP, and varying amounts of citric acid cycle intermediates or carnitine.
PYRUVATE
OXIDATION
IN MUSCLE
403
The rate of pyruvate oxidation is controlled by the availability of acetyl-CoA acceptor since addition of citric acid cycle intermediates or carnitine results in a stimulation of pyruvate oxidation. Pyruvate oxidation proceeds at its maximal rate in the presence of malate since no further stimulation is observed by addition of carnitine. It is concluded that pyruvate dehydrogenase is the rate-limiting step during pyruvate oxidation in the presence of malate. In human skeletal muscle mitochondria pyruvate oxidation proceeds maximally both in the presence of malate or carnitine. Parallel incubations of these mitochondria with [l-14Clpyruvate plus malate and [ l-14C] pyruvate plus carnitine may allow one to establish disturbances in pyruvate oxidation and to discriminate between defects in the pyruvate dehydrogenase complex and in the activity of the citric acid cycle. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
Linn, T. C., Pettit, F. H., and Reed, L. J., Proc. Nat. Acad. Sci. USA 62, 234 (1969). Wieland, 0.. and Siess, E., Proc. Nut. Acad. Sci. USA 65, 947 (1970). Hansford. R. G., J. Biol. Chem. 251, 5483 (1976). La Noue, K. F., Nicklas, W. J., and Williamson, J. R., J. Eiol. C&m. 245, 102 (1970). Schuster, S. M., and Olson, M. S., J. Viol. Chem. 247, 5088 (1972). La Noue, K. F., Bryla, J., and Williamson, J. R.. .I. Biol. Chem. 247, 667 (1972). Stucki. J. W., and Walter, P., Eur. J. Biochem. 30, 60 (1972). Dow, D. S., Biochemistry 6, 2915 (1967). Makinen, M. W., and Lee, C. P., Arch. Biochem. Biophys. 126, 75 (1%8). Hulsman, W. C., De Jong, J. W., and Van Tol, A., Biochim. Biophys. Acta 162, 292 (1%8). Peter, J. B., Biochem. Med. 2, 179 (1968). Bullock, G., Carter, E. E., and White, A. M. FEBS Letf. 8, 109 (1970). Max, S. R., Garbus, J., and Wehman. H. J., Anal. Biochem. 46, 576 (1972). Childress, C. C., Sacktor, B., and Traynor, D. R., J. Biol. Chem. 242, 754 (1966). Von Korff, R. W., and Kerpel-Fronius, S., J. Neurochem. 25, 767 (1975). Hansford, R. G., and Johnson, R. N., Camp. Biochem. Physjol. 55Jj, 543 (1976). Lysiak. W., Szutowicz, A., and Angielski, S., Acta Biochjm. Pa/on. 23, 325 (1976). Hutson. S. M., Van Dop, C.. and Lardy, H. A., J. Biol. Chem. 252, 1309 (1977). FOX, R. M., Anal. Biochem. 41, 578 (1971). Czok. R., and Lamprecht, W., “Methoden der enzymatischen Analyse” (H. U. Bergmeyer, Ed.), pp. 1491-14%. Verlag Chemie, Weinheim, 1974. Lowry, 0. H., Rosebrough, N. J., Farr. A. L., and Randall, R. J., J. Biol. Chem. 193, 265 (1951). Van Korff, R. W., Anal. Biochem. 8, 171 (1964). Pande, S. V., J. Biol. Chem. 246, 5384 (1971). Pande. S. V., Proc. Nat. Acad. Sci. USA 72, 883 (1975). Wieland, O., and Von Jagow-Westermann. B., FEES Lett. 3, 271 (1969). Fritz, 1. B.. and Yue, K. T. N., Amer. J. Physiol. 206, 531 (1964). Reiss, 0. K., and Hellerman, L., J. Biol. Chem. 231, 557 (1958). Robinson, B. H., Oei, J., Dhadli, S. C., and Halperin, M. L., J. Bio/. Chem, 252, 5661 (1977). Kark, R. A. P., Weinbank, E. C., Blass, J. P., and Engel, W. K., (1973) “Clinical Studies in Myology” (B. A. Kakulas, Ed.), pp. 98-107. Excerpta Medica Foundation. Amsterdam.
MEDICINE
20, 395-403
(I9781
Pyruvate Oxidation in Rat and Human Skeletal Muscle Mitochondria
A.J.
H. BOOKELMAN,* J. M. F. TRIJBELS,* R. C. A. SENGERS,* M. JANSSEN,* J. H. VEERKAMP,t AND A. M. STADHOLJDERS~ *Department of Pediutrics, tDepurtment of Biochemistr!. und SDepurtment of Submicroscopic Morphology. University qf Nijmegen. Nijmegen, The Netherlands Received
March
15, 1978
A vast amount of knowledge has accumulated during the past years regarding pyruvate oxidation. Regulation of the isolated enzyme as well as regulation of pyruvate oxidation in intact mitochondria have been s&died extensively (l-7). The majority of these studies has been performed with liver and heart muscle mitochondria. There have been hardly any studies concerning pyruvate oxidation in skeletal muscle mitochondria. The investigations performed so far are studies about isolation procedures and hence are only concerned with oxygen uptake and respiratory control (8-13). Pyruvate oxidation in mitochondria isolated from various tissues and species is known to be stimulated by citric acid cycle intermediates and carnitine (4,14-18). In this paper the stimulatory effects of citric acid cycle intermediates and carnitine on pyruvate oxidation in skeletal muscle mitochondria were studied in order to obtain information about the ratelimiting step during [I-r4C]pyruvate oxidation. The results obtained with rat skeletal muscle mitochondria are compared with those obtained with human skeletal muscle mitochondria, and their application for the diagnosis of a defect in human skeletal muscle pyruvate oxidation is discussed. MATERIALS AND METHODS Preparation qf skeletal muscle mitochondria. Rat skeletal muscle mitochondria were prepared from the hind leg of male Wistar rats, weighing 150-200 g and fasted overnight. Human skeletal muscle was obtained from patients with no known neuromuscular disease. The muscle specimens 395
0006-294417810203-0395$02.00/O CopyrIght @ 1978 by Academic Pres. Inc. All nghtc of reproduction in any form reserved
396
BOOKELMAN
ET AL.
were placed in ice-cold isolation medium containing 250 mM sucrose, 2 mM EDTA, 1OmM Tris, 50 IU of heparin/ml, final pH 7.4 (SETHmedium). All further operations were carried out at O-4”. Muscle tissue was freed from fat and connective tissue, cut into very fine pieces with scissors, and suspended in SETH-medium (1 g/10 ml). Homogenization was performed by hand with a Teflon-glass Potter-Elvehjem-type homogenizer. The resulting homogenate was centrifuged for 10 min at 6OOg to remove nuclei and cell debris. After repeating this centrifugation, mitochondria were isolated from the resulting supernatant by centrifugation for 10 min at 14,000g. The mitochondrial pellet was rinsed with SET-medium (which is SETH medium from which heparin is omitted) to remove the fluffy layer, resuspended in SET-medium, and centrifuged again for 10 min at 14,OOOg. The mitochondria were finally taken up in SET-medium. Oxygen consumption of the mitochondria, measured as described by Max et al. (13), showed respiratory control indices higher than 5, and P/O ratios between 2.8 and 3.0 with pyruvate and malate as substrates, indicating tight coupling between oxidation and phosphorybtion. Incubations and ussa~~. Mitochondria were incubated in small sealed glass vials at 37” in a shaking water bath. The basic incubation medium contained in a final volume of 500 ~1: 30 mM potassium phosphate, 2 mM EDTA. 10 mM Tris. 5 mM MgCl,, 75 mM KCI, 25 mrvt sucrose, 20 mrvr glucose, 2 mM ADP, and 1-2 units of hexokinase. The final pH was 7.4. Concentrations of substrates are given in the legends to figures and tables. Incubations were started by addition of 20-30 ,ug of mitochondrial protein. Small plastic tubes containing 200 ~1 of Hyamine were inserted into the vials in order to trap ‘*CO, produced during oxidation of [l-‘*Cl pyruvate (19). Incubations were terminated, after 10 or 15 min, by addition of 200 ~1 of 3 M HCIO,. The vials were left for an additional 30 min in the shaking water bath to ensure complete trapping of the 14C0, by the Hyamine. Thereafter, the small plastic tubes with the Hyamine were transferred into 20-ml glass counting vials containing 10 ml of toluene and 40 mg of Omnifluor. Radioactivity was measured in a Nuclear Chicago Mark 1 liquid scintillation counter. Under all conditions tested, ‘4c0, production from [I-Wlpyruvate was linear with respect to time and the amount of protein present. Pyruvate was determined enzymatically according to Czok and Lamprecht (20). Protein was determined by the method of Lowry et al. (21) using bovine serum albumin as a standard. Statistical analyses were performed with Student’s t test. Chemicals. The sodium salt of [ 1-‘*C]pyruvate was purchased from the Radiochemical Centre, Amersham, England, and stored freeze-dried under nitrogen in sealed ampoules at -20” to minimize decomposition (22). Further reagents were purchased as follows: L(--)-carnitine and
PYRUVATE
OXIDATION
397
IN MUSCLE
hexokinase (freeze-dried, 40 units/mg) from Koch Light, Colnbrook. gland: rotenone and ADP from Boehringer und Sohne, Mannheim, Germany; Omnifluor from NEN chemicals GmbH, Frankfurt, West many; and the hydroxide of Hyamine 10X from Packard Instrument Chicago, Ill., U.S.A. All other chemicals were of the purest grade mercially available.
EnWest GerCo.. com-
RESULTS Stimulation of pyruvate oxidation by citric acid cycle intermediates. The effect of increasing concentrations of malate, fumarate, and succinate on the rate of pyruvate oxidation by rat skeletal muscle mitochondria is shown in Fig. 1. All the citric acid cycle intermediates tested stimulate pyruvate oxidation, although the effect of fumarate and succinate is smaller than that observed with malate. In all instances a final concentration of 1 mM of the added citric acid cycle intermediates is enough to approximate the maximal stimulatory effect which can be obtained with a particular intermediate. In the presence of malate the rate of pyruvate oxidation measured by the production of 14C0, from [l-14C]pyruvate equals the rate of pyruvate
-,-
”
0.5 cltrlc
acid
1.0 cycle
2.0 intermedIate
[mM]
FIG. 1. Stimulation of pyruvate oxidation by citric acid cycle intermediates. Rat skeletal muscle mitochondria (54 cog of protein/ml) were incubated in the basic incubation medium supplemented with I mM pyruvate (0.15 &i of [I-‘4Clpyruvate). Malate, fumarate, and succinate were present in final concentrations shown in the figure. 0-O. malate: X-X. fumarate: O-0, succinate.
398
BOOKELMAN
ET AL.
disappearance from the incubation medium measured enzymatically, indicating that all the pyruvate taken up by the mitochondria is oxidized to CO, (Table 1). Stirnulution of pynrvcrte oxidation by cnrnitine. The rate of 14C0, production from [l-‘“Clpyruvate is not only increased by addition of citric acid cycle intermediates but also by addition of carnitine to the mitochondria. Acetyl-CoA formed by the pyruvate dehydrogenase complex can be transferred to carnitine by the action of carnitine acetyltransferase (23,241 to form acetylcarnitine and to regenerate mitochondrial CoASH, thereby relieving inhibition of the pyruvate dehydrogenase complex by acetylCoA (25). Carnitine acetyltransferase activity has been shown to be present in muscle mitochondria (26). The effect of carnitine on pyruvate oxidation can be seen in Fig. 2. The double reciprocal plot of oxidation rate versus carnitine concentration shows biphasic kinetics. Two apparent K,, values of 0.2 m and 3.7 mM can be calculated corresponding with a V max of 4.1 and 16.1 pmoles/mg of protein .hr, respectively. The V,,, reached at infinite carnitine concentration approximates closely the oxidation rate obtained in the presence of I mM malate which was 17.0 pmoles/mg of protein.hr in this experiment. In the presence of malate no acetate formation was observed. The main products formed in the presence of carnitine were acetylcarnitine and acetate, the latter probably resulting from hydrolysis of acetylcarnitine. Pynrvcrte oxidcltiotl ,cithout addition of citric acid cycle intermediates or camitine. An attempt was made to investigate whether the low rate of pyruvate oxidation measured when no citric acid cycle intermediates or carnitine were added was due to pyruvate oxidation supported by endogenous citric acid cycle intermediates or due to some nonspecific decarboxylation of the [ I-%]pyruvate. Therefore, the effect of arsenite, an inhibitor of the pyruvate dehydrogenase complex (27) was studied. Figure 3 shows the effect of arsenite on pyruvate oxidation measured without TABLE RATE
Parameter Pyruvate Pyruvate
OF PYRUVATE
UPTAKE
I AND
Rate (pmoles/mg uptake oxidation
OXIDATION”
of protein
.hr)
16.3 t 1.9 (4) 15.8 2 1.2 (6)
I’ Rat skeletal muscle mitochondria were incubated in the basic incubation medium supplemented with 1 mM pyruvate (0.15 &i of [l-W] pyruvate) and 1 mM malate. Pyruvate uptake was measured as the difference in pyruvate concentration in the neutralized deproteinized perchloric acid extracts of the medium before and after incubation. Pyruvate oxidation was measured as described in Materials and Methods. Values shown are means ? SD of the number of experiments given in parentheses.
PYRUVATE
OXIDATION
399
IN MUSCLE
0
i
i 1
[carnltlne] FIG. 2. Lineweaver-But-k skeletal muscle mitochondria. basic incubation medium.
4 mM-’
plot for carnitine stimulation of pyruvate oxidation in rat Pyruvate ( ImM) (0. I5 &i of [I-‘4Clpyruvate) was added to the
additions, with carnitine and with malate. A prompt inhibition of pyruvate oxidation by low concentrations of arsenite is observed in the presence of malate or carnitine. However, virtually no inhibition is observed when pyruvate oxidation is measured without addition of citric acid cycle intermediate or carnitine. Moreover, the residual activity measured under all three conditions is the same. This indicates that part of the pyruvate
161
0
01 arsenate
0.2 [ mM I
FIG. 3. Inhibition of pyruvate oxidation by arsenite. Rat skeletal muscle mitochondria (115 fig of protein/ml) were incubated in the basic incubation medium supplemented with 1 mM pyruvate (0.15 &i of [I-‘4Clpyruvate). Further additions were: O-0, none: O-O, 5 mM Carnitine; X-X, 1 mM malate.
400
BOOKELMAN
ET AL.
TABLE EFFECT
OF INHIBITORY CITRIC
ON PYRUVATE ACID
CYCLE
2
OXIDATION
IN THE
INTERMEDIATES
ABSENCE
OF ADDED
OR CARNITINE”
Addition
Pyruvate oxidation rate (pmoles/mg of protein. hr)
None Rotenone Malonate Arsenite
1.5 0.2 0.5 1.1
” Rat skeletal muscle mitochondria (46 &ml) were incubated in the basic incubation medium supplemented with 1 mM pyruvate (0.15 &i of [I-‘Qpyruvate). Incubations were terminated after 15 min. Inhibitor concentrations were I-&ml rotenon, 5 mM malonate, 0.2 mM arsenite.
oxidation measured is insensitive to arsenite inhibition and hence this activity may not represent pyruvate dehydrogenase activity. This conclusion is not supported by the effects observed with rotenone or malonate as shown in Table 2. The residual activity is completely destroyed by heating of the mitochondria (3 min at 100’) prior to incubation. Pyruvate oxidation in rat and human skeletal muscle mitochondria. In Table 3 the stimulatory effects on pyruvate oxidation of the citric acid TABLE EFFECT
OF CITRIC
ACID
PYRUVATE
CYCLE
3
INTERMEDIATES
OXIDATION
IN RAT
MUSCLE
AND AND
CARNITINE
HUMAN
ON THE
RATE
OF
SKELETAL
MITOCHONDRIA”
Pyruvate oxidation rate
Addition
Rat (pmoles/mg of protein .hr)
Human (~moles/mg of protein .hr)
None Malate Fumarate Succinate Carnitine Carnitine + malate Carnitine + fumarate Carnitine + succinate
1.1 15.8 7.5 10.7 8.3 16.4 13.0 14.7
2.0 7.5 3.0 4.9 7.9 8.8 7.1 7.3
2 T 2 2 2 2 f 2
0.2 (5) 1.2 (6) 1.1 (4) 0.6 (4) 0.8*(5) 1.7 (5) 1.9*(4) 1.4*(4)
of 0.8 (5) + 3.5 (6) 2 1.3 (4) * 2.0 (4) k 3.8*(6) 2 4.3 (5) z!r 3.3 (4) 2 3.8 (4)
Relative (%) 25? 100 45 It 732 105 * IlOk 106 ” 107 t
3 10 5 9* 7 12* 9*
4 Mitochondria were incubated in the basic incubation medium supplemented with I mM pyruvate (0.15 &i of [l-‘Flpyruvate). Additions of citric acid cycle intermediates (1 mM) or carnitine (5 mM) were made as indicated. Values shown are means * SD of the number of experiments given in the parentheses. The significance of the difference in oxidation rate in the presence and absence of carnitine was calculated. * P < 0.05.
PYRUVATE
OXIDATION
IN MUSCLE
401
cycle intermediates malate, fumarate, and succinate are compared with the effect of carnitine in rat and human skeletal muscle mitochondria. The values for the rate of pyruvate oxidation obtained with human skeletal muscle mitochondria are presented in two manners. Both the absolute values (pmoles/mg of protein *hr) and the relative values (expressed as percentage of those obtained with 1 mrvr malate) are presented. In the latter manner standard deviations were greatly reduced. Both in rat and human skeletal muscle mitochondria maximal stimulation of pyruvate oxidation is achieved by malate although this effect is greater in rat than in human skeletal muscle mitochondria. A decrease in stimulatory effect in the order malate> succinate> fumarate is observed both with rat and human muscle mitochondria. Addition of carnitine results in a stimulation of pyruvate oxidation rate which is in rat skeletal muscle mitochondria half of that observed with malate while in human skeletal muscle mitochondria the same stimulation is achieved as with malate. In skeletal muscle mitochondria of both species pyruvate oxidation measured in the presence of fumarate or succinate can be further stimulated by addition of carnitine. In the presence of malate no further stimulation by carnitine is observed. DISCUSSION Pyruvate oxidation in skeletal muscle mitochondria incubated with I mrvf pyruvate and an excess of ADP but without addition of a citric acid cycle intermediate or carnitine is limited by the availability of oxaloacetate. This can be concluded from the stimulatory effect of citric acid cycle intermediates and carnitine. The differences which are observed between the stimulatory effects of malate, fumarate, and succinate (Fig. 1) are probably caused by differences in the permeability of the mitochondrial membrane toward these intermediates. The role of acceptor for acetyl-CoA generated by the action of the pyruvate dehydrogenase complex is not restricted to oxaloacetate but can also be fullfilled by carnitine (14, 16, 18, 23, 24). Ths biphasic kinetics of the stimulation of pyruvate oxidation by carnitine shown in Fig. 2 points to the action of two different enzymes. The V,,, reached at infinite carnitine concentration closely approximates the activity found in the presence of malate. So, both malate and carnitine are able to stimulate pyruvate oxidation to the same extent although the effect of malate is reached at far lower concentrations. Pyruvate oxidation measured without addition of citric acid cycle intermediate or carnitine is supported by endogenous citric acid cycle intermediates. This can be concluded from the observed inhibition by malonate (Table 2). Spontaneous decarboxylation cannot be responsible for this residual activity since the latter is inhibited by malonate,
402
BOOKELMAN
ET AL.
rotenone. and heating of the mitochondria. The partial insensitivity toward arsenite was attributed in a recent report concerning kidney mitochondria (28) to an overestimation of the amount of evolved 14C0,. However, since arsenite is a water-soluble reagent and the pyruvate dehydrogenase complex is bound to the inner mitochondrial membrane, partial inaccessibility of the pyruvate dehydrogenase complex toward arsenite in intact mitochondria is an alternative explanation. Citric acid cycle intermediates and carnitine stimulate pyruvate oxidation both in rat and human skeletal muscle mitochondria. Large standard deviations were obtained with human skeletal muscle mitochondria when the activities are expressed on protein base (Table 3). This was reported previously (29). Therefore, the problem of finding a better reference base to express the activities found in human material needs further investigation. The stimulatory effect of carnitine on pyruvate oxidation in the presence of fumarate or succinate indicates that with these two citric acid cycle intermediates the availability of the acetyl-CoA acceptor, oxaloacetate, is still rate-limiting for maximal pyruvate dehydrogenase activity. Since no further stimulation of pyruvate oxidation can be achieved by carnitine in the presence of malate, the rate of acetyl-CoA production is the rate-limiting step during [I-‘4C]pyruvate oxidation under this condition. The stimulation of pyruvate oxidation by carnitine is equal to that observed with malate in human skeletal muscle mitochondria in contrast to that in rat skeletal muscle mitochondria. This suggests that carnitine acetyltransferase activity is relatively greater in human skeletal muscle mitochondria. Parallel incubations of human skeletal muscle mitochondria with [ I-‘“Clpyruvate plus malate or carnitine will be the best means of obtaining information about the capacity of human skeletal muscle mitochondria to oxidize pyruvate. It not only permits the diagnosis of an impaired rate of pyruvate oxidation, but it also discriminates between a defect in pyruvate dehydrogenase and an impaired capacity of the mitochondria to oxidize acetyl-CoA via the citric acid cycle. In case of a defect in the respiratory chain, it can be expected that pyruvate oxidation will be impaired both with malate and with carnitine added. Then further investigations are indicated to determine whether the defect in pyruvate oxidation is due to a defect in pyruvate dehydrogenase or whether the defect is secondary to a defect in the respiratory chain. SUMMARY
Pyruvate oxidation in rat and human skeletal muscle mitochondria was studied by measuring the rate of ‘CO, production from [lJ4C]pyruvate in the presence of 1 mM pyruvate, an excess of ADP, and varying amounts of citric acid cycle intermediates or carnitine.
PYRUVATE
OXIDATION
IN MUSCLE
403
The rate of pyruvate oxidation is controlled by the availability of acetyl-CoA acceptor since addition of citric acid cycle intermediates or carnitine results in a stimulation of pyruvate oxidation. Pyruvate oxidation proceeds at its maximal rate in the presence of malate since no further stimulation is observed by addition of carnitine. It is concluded that pyruvate dehydrogenase is the rate-limiting step during pyruvate oxidation in the presence of malate. In human skeletal muscle mitochondria pyruvate oxidation proceeds maximally both in the presence of malate or carnitine. Parallel incubations of these mitochondria with [l-14Clpyruvate plus malate and [ l-14C] pyruvate plus carnitine may allow one to establish disturbances in pyruvate oxidation and to discriminate between defects in the pyruvate dehydrogenase complex and in the activity of the citric acid cycle. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
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