- Email: [email protected]
Metabolism of pyruvate and carnitine esters in bovine epididymal sperm mitochondria
ARCHIVES
OF
BIOCHEMISTRY
Metabolism
AND
BIOPHYSICS
of Pyruvate
for Enzyme
Research
and
345-352
(1977)
and Carnitine Esters in Bovine Sperm Mitochond rial
S. M. HUTSON, Institute
181,
C. VAN DOP,2
AND
the Department of Biochemistry, Wisconsin 53706 Received
December
Epididymal
H. A. LARDY University
of Wisconsin,
Madison,
6, 1976
Filipin-treated bovine epididymal spermatozoa have been used to study mitochondrial L-acetylcarnitine, t-palmitoylcarnitine, and pyruvate metabolism. The cells were supplemented with malatc to allow rapid rates of substrate oxidation. The rate of Lpahnitoylcamitine-supported state 3 respiration was slow. In contrast, pyruvate, acetylcarnitine, or lactate supported rapid and approximately equal respiratory rates. LPalmitoylcarnitine was a weak inhibitor of pyruvate-supported respiration and pyruvate use and a more potent inhibitor of L-acetylcarnitine. L-Camitine was an effective inhibitor of L-acetylcamitine oxidation; however, it did not influence L-palmitoylcarnitine oxidation or inhibit pyruvate utilization. Pyruvate (1.4 mM) disappearance was rapid and was complete within 6-7 min; the lactate produced during pyruvate metabolism was then oxidized. ATP synthesis was constant throughout the 20-min incubation. With pyruvate plus L-acetylcamitine as substrate, the L-acetylcarnitine concentration initially dropped and then recovered to a level that was dependent on free camitine addition. Data obtained from experiments using [2-14C]pyruvate indicated that the 14C label from pyruvate and lactate entered the L-acetylcamitine pool and labeling was maximal when free n-camitine was added. The rate of citrate synthesis was maximal when pyruvate was being metabolized; the largest total accumulation occurred when all three substrates were included in the incubation. The data suggest that the high NAD+/ NADH maintained during pyruvate metabolism may restrict flux through the citric acid cycle. The relationships of L-camitine and the L-camitine esters to pyruvate metabolism are discussed.
In mammalian sperm, the intramitochondrial location of lactate dehydrogenase-X (l-3) allows pyruvate to oxidize the NADH produced from its own metabolism (4, 5). Metabolism of pyruvate in these cells is exclusively intramitochondrial (4, 5). Recent reports (4,6,7) have also shown that bovine spermatozoa oxidizing pyruvate maintain high L-acetylcarnitine:carnitine ratios. The effects of L-carnitine and L-acetylcarnitine on pyruvate metabolism in heart and blowfly flight muscle mitochondria have been well documented (8-10). Casillas (11) has shown that in sonicated 1 Supported by Grants AM 10,334 and HD from the National Institutes of Health. 2 Recipient of Training Grant No. 2T05-GM from the National Institutes of Health.
spermatozoa, L-carnitine enhances the rate of palmitate oxidation. However, in ejaculated bovine spermatozoa, the addition of L-carnitine depressed fatty acid oxidation and stimulated incorporation of palmitate into 1,kdiglycerides (12). Bovine epididymal spermatozoa can oxidize endogenous lipids (13), but, in hypotonically treated rabbit epididymal spermatozoa, Lpalmitoylcarnitine oxidation was slow (14). Recently, we have used the polyene antibiotic, filipin, to study mitochondrial metabolism in bovine epididymal spermatozoa (5). Filipin renders the spermatozoan cell membrane permeable to small molecules (15). L-Carnitine addition to filipintreated spermatozoa metabolizing pyruvate resulted in a linear rate of cacetylcarnitine synthesis, which was maximal
08630 01932 345
Copyright All rights
0 1977 hy Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
346
HUTSON,
VAN DOP, AND LARDY
when oxalacetate was limiting (no added malate). The purpose of the present study was to examine the relationship(s) between metabolism of L-acetylcarnitine, long-chain acylcarnitine, and pyruvate. In addition, the effects of L-carnitine on the metabolism of these substrates have been examined. MATERIALS AND METHODS Preparation and treatment of bovine spermatozoa. Preparation, filipin treatment, and incubation of the bovine epididymal spermatozoa were as previously described (5). The standard incubation medium contained 15 mM Tris-phosphate, 10 mM MgSO,, 0.5 mM Tris-ADP, 35 mM 2-deoxyglucose, 0.1 mglml of hexokinase (dialyzed vs 15 mM Trisphosphate, pH 7.4, immediately before use), 250 mM mannitol, and 15 mM Tris(Cl), pH 7.4. When added, carnitine (pH 7.4, Tris) was 50 mM (5). All experiments were conducted at 37°C. Measurement of respiratory rates. Respiratory rates were measured using a Clark electrode attached to a Gilson oxygraph (Gilson Medical Electronics, Middleton, Wisconsin (5)). Radioactive experiments. Conversion of pyruvate to other metabolites was followed by incubating sperm suspensions (volume, 1.0 ml) in stoppered scintillation vials with 1.4 mM pyruvate containing 0.6 to 1.0 &i of [2X!lpyruvate. The concentrations of all stock substrate solutions were measured ensymatically. “C-labeled metabolites were separated by the chromatographic procedure of LaNoue et al. (16). Details of the incubation procedure and chro-
matographic separation pattern for tilipin-treated bovine epididymal sperm have been described (51. Assay methods. Pyruvate, lactate, malate, free Lcarnitine, L-acetylcarnitine, long-chain acylcarnitine, 2-deoxyglucose g-phosphate, glutamate, citrate, aspartate, /3-hydroxybutyrate, and fumarate were measured using standard techniques (4, 5). Materials. The sources of bovine epididymides, enzymes, L-carnitine, filipin, substrates, and [2‘Qpyruvate were as previously described (4, 5). LAcetylcarnitine and L-palmitoylcarnitine were obtained from P-L Biochemicals. RESULTS
Effects of the Various Substrates on Sperm Respiration
Malate (0.8 mM) did not support a significant rate of respiration but was included in all of the incubations because it was necessary for significant rates of palmitoylcarnitine or acetylcarnitine oxidation. cPalmitoylcarnitine-supported state 3 (ADP present) respiration was slow (Fig. 1A). Concentrations of L-pahnitoylcarnitine of from 12.5 to 200 PM supported maximal rates of respiration; at 400-500 PM, the surfactant effect was significant and respiration was inhibited. Unlike palmitoyl-CoA, palmitoylcarnitine is not a potent inhibitor of the adenine nucleotide translocase (17, 18). Sperm respiration supported by pyruvate or L-acetylcarnitine was inhibited by
FIG. 1. Effects of the various substrates on sperm respiration. Sperm were incubated in the standard incubation medium as described under Materials and Methods. The sperm concentrations were 2.8 x lOa (A-C) and 1.8 x 10s (D) cells/ml. Pyruvate and L-acetylcarnitine were 1.30 and 1.75 mxr, respectively. Malate (0.77 mM) was included in all incubations. The numbers in parentheses are the respiratory rates expressed as nanogram atoms of oxygen per 10s cells per minute.
METABOLISM
OF SPERM MITOCHONDBIA
increasing concentrations of L-palmitoylcarnitine (Figs. 1B and 10 With pyruvate as the substrate, 100 PM palmitoylcarnitine (the concentration used in subsequent metabolite experiments) inhibited respiration about 20%. L-Carnitine completely reversed the inhibitory effect of Lpalmitoylcarnitine on pyruvate oxidation (data not shown). With L-acetylcarnitine as substrate, 100 PM L-palmitoylcarnitine inhibited respiration significantly (Fig. 10. In contrast to the case with pyruvate (51, respiration supported by L-acetylcarnitine was progressively depressed by increasing concentrations of L-carnitine (Fig. 1D). Maximal inhibition was 75%. rEarmtine inhibits ~acetylcarnitine oxidation in blowfly flight muscle mitochondria (10, 19). In hypotonically treated rabbit spermatozoa, the L-acetylcarnitine-supported respiratory rate was about 25% of that of pyruvate, and L-acetylcarnitine inhibited the rate of pyruvate oxidation (14). In filipin-treated bovine epididymal sperm, Lacetylcarnitine was oxidized as well as pyruvate and had no effect on the pyruvatesupported respiratory rate. In other experiments (data not shown), lactate supported a respiratory rate equal to that with pyruvate. In combination with pyruvate, the respiratory rate was about the same as with either substrate alone. L-Acetylcarnitine and Pyruvate Metabolism The state 3 rate (ADP present) of Lacetylcarnitine-malate metabolism in filipin-treated spermatozoa is shown in Fig. 2A. GAcetylcarnitine disappearance was always faster during the first 4 min than during the remainder of the incubation [0.042 -+ 0.017 (4) and 0.130 f 0.002 (3) pmol used/loH cells at 4 and 20 min, respectively]. Malate disappearance always paralleled citrate synthesis and is usually not included in subsequent figures. As shown in Fig. 2B, pyruvate addition to cells metabolizing L-acetylcarnitine largely blocked Gacetylcarnitine disappearance. After an initial drop, n-acetylcarnitine remained at 85% of its initial
347
FIG. 2. Comparison of sperm mitochondrial Lacetylcarnitine and pyruvate metabolism. Twicewashed iilipin-treated cells were incubated in the standard incubation medium (see Materials and Methods). The sperm concentration was 9.9 x 10’ cells/ml. Substrate additions were as follows: PyNvate, 1.64 mM; cacetylcarnitine, 2.32 m&r; malate, 0.77 mM. The symbols used are: L-acetylcarnitine (a); malate (0); citrate (A); pyruvate (W); lactate (0).
concentration throughout the rest of the 20-min incubation. At the pyruvate and cell concentrations used in these experiments (1.4 mM and 6-10 x 10s cells/ml, respectively), the rate of pyruvate disappearance was linear and complete within 6-7 min after pyruvate addition (see Fig. 3). In agreement with previous reports concerning heart and blowfly flight muscle mitochondria (9, lo), L-acetylcarnitine addition did not affect the rate of pyruvate use (compare Figs. 2B and 20. Lactate appeared to be metabolized in preference to L-acetylcarnitine after all of the pyruvate had been used (Figs. 1B and 10. It seems likely that lactate is being oxidized via lactate dehydrogenase-X and pyruvate dehydrogenase; some of the acetate is shunted into acetylcarnitine and the rest is being oxidized via the citric acid cycle. In Figs. 2B and 2@, at 4 min, citrate accumulation was already 50% of the final value at 20 min; thus, citrate synthesis was maximal when pyruvate was being metabolized. The total amount of citrate that accumulated was largest in the presence af both substrates (Fig. 2B). Effects of L-Palmitoylcarnitine L-Palmitoylcarnitine (0.17 mM) inhibits the decarboxylation of pyruvate in rat
348
HUTSON,
VAN
heart mitochondria (9). L-Palmitoylcarnitine (100 PM) was a very weak inhibitor of pyruvate metabolism (Figs. 3A and 3B); in other experiments, maximal inhibition of the rate of pyruvate use was never greater than 20%. As in rat heart (9), pyruvate significantly inhibited L-palmitoylcarnitine disappearance (compare Figs. 3B and 3C). Pyruvate inhibition of L-palmitoylcarnitine metabolism was not further characterized, because the long-chain acylcarnitine extraction procedure was unreliable. The effects of L-palmitoylcarnitine on Lacetylcarnitine and pyruvate metabolism are illustrated in Figs. 4A-4C. The corresponding controls are shown in Fig. 2. LPalmitoylcarnitine inhibited cacetylcarnitine disappearance by about 35% (Fig. 4A); in other experiments, the inhibition ranged from 35-55%. Direct comparison of the data obtained in the respiration and metabolism experiments is not possible, because cell concentrations were two to four times greater in the experiments in which metabolites were measured. Citrate synthesis was increased by palmitoylcarnitine and was maximal with all three substrates present (Fig. 40 When all three substrates were included in the incubation, L-acetylcarnitine initially decreased was then reacetylated to 94% of its original concentration (Fig. 40.
FIG. in Fig. malate lactate
DOP,
AND
Effects
LARDY
of L-Carnitine
rK!arnitine has a significant function in carbohydrate metabolism in bovine epididymal sperm (4, 6, 7) and filipin-treated sperm (5). In the filipin-treated sperm, Lcarnitine addition to cells metabolizing Lacetylcarnitine inhibited acetylcarnitine use about 50%, with a concomitant decrease in malate use and citrate synthesis (Fig. 5A). In contrast to the data obtained with acetylcarnitine, carnitine did not influence L-palmitoylcarnitine oxidation (data not shown). The corresponding controls without L-carnitine are shown in Fig. 2. As shown previously, L-carnitine did not alter the rate of pyruvate use, but consistently decreased the amount of pyruvate that was converted to lactate by 5-10%; part of the pyruvate was preferentially shunted into L-acetylcarnitine (Table I and Ref. 5). The data in Figs. 5B and 5C suggest that lactate must have been a source of acetyl-CoA for carnitine acetyltransferase. rCarnitine addition also reduced citrate accumulation (compare Figs. 2 and 5). In other experiments (data not shown), Lcarnitine eliminated any effects of ~-palmitoylcarnitine on pyruvate use. Metabolism
The
of Malate and [2-WPyruvate
metabolism
of malate
and
3. Effect of L-palmitoylcarnitine on pyruvate metabolism. Conditions were the same as 1. The sperm concentration was 1.1 x lo@ cells/ml. F’yruvate was added at 1.36 mM, at 0.77 mM, and L-palmitoylcarnitine at 100 pM. The symbols used are: pyruvate (a); (0); net free carnitine (A).
[2-
METABOLISM
OF SPERM
FIG. 4. Metabolism of L-acetylcamitine, L-palmitoylcamitine, and pyruvate. Incubation conditions and cell and substrate concentrations were the same as in Fig. 2; the symbols are the same as in Fig. 2. In addition, L-palmitoylcarnitine was 100 PM.
MITOCHONDRIA
349
In general, label did not pass through the citric acid cycle more than once. Similarly, the malate pool was not replenished with malate containing 14C. Even though greater than 80% of the malate had been used by 20 min, the final specific activity of the remaining malate ranged from 20 to 30% of the initial pyruvate. Total fumarate and aspartate were less than 10 nmol/108 cells at any time point. Glutamate was about 10 nmol/lOs cells and did not vary under any conditions tested. Free acetoacetate and /3-hydroxybutyrate were not detectable. Oxidative Phosphorylation
The synthesis of 2-deoxyglucose B-phos14C]pyruvate is summarized in Tables I phate was used as a measure of ATP production (15), but remains a qualitative and II. The release of ‘4c02 was low and measure because of ATPase activity (5). even after 20 min was less than 20% of the ATP synthesis in cells oxidizing acetylcartotal pyruvate added. As in blowfly flight the incubamuscle mitochondria (3), L-carnitine ap- nitine was linear throughout tion period (Fig. 6-1) and the average of peared to decrease 14C02 release; but the percentage was quite variable. Less 14C four values at 20 min was 0.653 + 0.064 ~mol/lO* cells. Although L-palmitoylcarnilabel accumulated in COz and succinate tine inhibited acetylcarnitine disappearwhen unlabeled L-acetylcarnitine and Lance, this was not reflected in the rate of palmitoylcarnitine were added. At 20 min, oxidative phosphorylation (Fig. 6-2). Genthe ‘4c label had accumulated in most citerally, the effects of the various comric acid cycle intermediates. However, pounds on the rate of oxidative phosphomost of the 14C label was concentrated in rylation supported by acetylcarnitine relactate, citrate, and, with L-carnitine, in Lflected the results seen in the respiration acetylcarnitine as well. and metabolism experiments. In cells oxiThe data indicate that label from lactate entered the L-acetylcarnitine pool (Table I, Fig. 2), for pyruvate had disappeared during the first 6-7 min and acetylcarnitine continued to accumulate ‘4c label. With all three substrates plus carnitine, there was net acetylcarnitine synthesis and the largest 14C labeling. The fractions containing L-acetylcarnitine were analyzed after alkaline hydrolysis (Table II). If a source of carnitine was available, acetate comprised 8595% of the labeled material; without carnitine, acetate comprised about 45%. An unknown component, tentatively identified as lactate or /3-hydroxyFIG. 5. Effects of L-carnitine on L-acetylcarnitine butyrate (8, ranged from 2-5 nmol/108 and pyruvate metabolism. Conditions were the cells, while the unhydrolyzed compound same as in Fig. 2; the symbols are the same as in (probably alanine) (16) was constant at Fig. 2. The sperm concentration was 7.8 x lo* cells/ about 1.5 nmol. ml. L-Camitine was 50 mM, malate was 0.96 mM The specific activity of the [14C]citrate and, when present, pyruvate was 1.51 mM. L-Acetylwas similar to that of the [2-14C]pyruvate. camitine was 2.05 mM.
350
HUTSON,
VAN DOP, AND LABDY TABLE
EFFECT
I
OF L-CARNITINE, GACET~LCARNITINE, AND L-PALMITOYLCARNITINE ON THE 14C! LABELING METABOLITES IN SPERM OXIDIZING MALATE AND [2-14C]Py~w~~~a
Other additions
None L-Camitine L-Acetylcamitine + L-palmitoylcamitine L-Acetylcarnitine + L-palmitoylcamitine + L-camitine
OF
Sampp;t (min)
Pyruvate metabolizedb
I Acetylcamitine”
II Acetate glutamate6
III Lactat&
VIII Citrate6
v succinate”
4 20 20 4 20 20
135 189 157 81 161 249
9 9 10 11 52 42 (203)”
3.5 14 17 4 12 32
74 50 55 39 33 99
25 50 35 26 30 60
9 10 8 3
3 19 9 12 2
7 38 21 2 19 10
20
236
79 (325)
32
41
82
2
3
6
IbEate*
14coI*
a Conditions are the same as in Fig. 1. The sperm concentration ranged from 6.2 to 8.1 x lOa cells/ml. Pyruvate ranged from 1.1 to 1.44 mM and malate was 0.8 mM. Initial r.-acetylcamitine was 1.72 mM (275 nmol/lO* cells) and L-palmitoylcamitine was 100 pM. At 20 min, 99% of the initial pyruvate had been used. Roman numerals heading six of the columns designate the chromatographic fraction and the major component(s) of the fraction. * Nanomoles per lo* cells, based on the specific activity of the [2-“Clpyruvate. c The enzymatically determined concentration of acetyl camitine is given in parentheses. TABLE ANALYSIS
II
OF THE ALKALINE COMPONENT
Additions
HYDRQLYSATE
Ia Unhydrolyzedb 1.8 1.3 1.0
Acet&e*
OF
Unknown”
None 4.1 3.9 L-Camitine 50.2 1.2 n-Acetylcarnitine 35.4 5.4 + L-palmitoylcarnitine 1.3 76.1 2.0 L-Acetylcarnitine + L-palmitoylcamitine + L-carnitine n The pooled fractions from Peak I (see Table I) were rechromatographed on Dowex I after alkaline hydrolysis. b Nanomoles per lo8 cells per 20 min; calculations are based on the specific activity of the [2“Clpyruvate.
dizing palmitoylcarnitine, ATP synthesis was slow and was not influenced by carnitine (Fig. 6-4). However, carnitine was inhibitory in cells oxidizing acetylcarnitine (Fig. 6-5); further addition of cpalmitoylcarnitine resulted in an intermediate rate of synthesis (Fig. 6-3). Thus, while acetylcarnitine oxidation was inhibited by carnitine, palmitoylcarnitine oxidation was unaffected. The rate of 2-deoxyglucose 6-phosphate
synthesis in cells metabolizing pyruvate was constant for 15 to 20 min (Fig. 6-6). G Carnitine, L-acetylcarnitine (Fig. 6-71, or L-palmitoylcarnitine, or combinations of these substrates, did not have any effect on this rate. The typical variation within a single experiment was 2-8%; among different cell populations, the variation was approximately 15%. DISCUSSION
The data presented here confrm earlier observations made on intact cells (4) which suggested that lactate was not metabolized when pyruvate was present. However, after all of the pyruvate was used, lactate was metabolized. Lactate use was much slower than pyruvate use, but lactate-supported respiration was equal to that with pyruvate. Storey and Kayne (20) have reported that, in hypotonically treated rabbit epididymal sperm, lactate was oxidized more rapidly than pyruvate. This difference may be the result of the differing treatments or species. ~Acetylcarnitine or lactate plus malate gave comparable reduction of mitochondrial pyridine nucleotides (20). Spermatozoa metabolizing pyruvate maintain a high NAD+/NADH (21, 22). Data presented here and earlier work
METABOLISM
0
5
I5
OF SPERM
20
FIG. 6. Effects of the various treatments on 2deoxyglucose 6-phosphate synthesis. Incubation conditions and substrate and cell concentrations were the same as in Figs. 2 and 4. Malate was included in all incubations, Cells were metabolizing the following substrates: L-acetylcarnitine (11, Lacetylcarnitine plus L-palmitoylcarnitine (21, both substrates plus L-carnitine (31, L-palmitoylcarnitine plus L-carnitine (41, n-acetylcamitine plus L-carnitine (51, pyruvate (61, and pyruvate plus L-acetylcarnitine (7).
(5) suggested that in filipin-treated cells pyruvate is incompletely oxidized. During pyruvate metabolism, citrate accumulation was large and 14C02 release was low. Furthermore, the data imply that lactate, and probably tacetylcarnitine, are completely oxidized via the citric acid cycle. This leads directly to the possibility that when the mitochondrial pyridine nucleotides are highly oxidized, flux through the citric acid cycle is limiting for pyruvate oxidation. The block may be at isocitrate is supdeh ydrogenase . This postulate ported by the pattern of citrate accumulation. Control of flux through the citric acid cycle in sperm may be very different from that seen in heart mitochondria (16, 23, 24).
Ever-se and Kaplan (25) proposed that under normal in uiuo conditions, the Htype lactate dehydrogenase is prevented from reducing pyruvate to lactate, while maintaining its full capacity to oxidize lactate. A high NAD+/NADH and a high pyruvate/lactate would favor abortive complex formation; lowering the pyruvate/lac-
MITOCHONDRIA
351
tate would favor oxidation of lactate. Certain kinetic properties of lactate dehydrogenase-X, in particular the striking inhibition by pyruvate, have led Blanco et al. (3) to extend this hypothesis to the sperm enzyme. Lactate dehydrogenase-X represents 80% or more of the total lactate dehydrogenase activity of spermatozoa (26). In sperm mitochondria, pyruvate was preferentially reduced to lactate. As shown previously (41, pyruvate reduction occurred over a wide range of pyruvate/lactate even with a high NAD+/NADH. Thus, the mitochondrial NAD+/NADH ratio may be the most important regulatory parameter for lactate dehydrogenase-X. Casillas and Erickson (6) postulated that, in spermatozoa, the acetylcarnitine:carnitine transferase system is used to buffer against rapid changes in the concentration of acetyl-CoA. More recently, Milkowski et al. (7) and Van Dop et al. (4) have suggested that L-acetylcarnitine may also serve as a source of “active” acetate. Childress et al. (10) postulated a similar role for acetylcarnitine in insect flight muscle (under oxalacetate-limiting conditions). With pyruvate and L-acetylcarnitine as the substrates, the L-acetylcarnitine concentration initially dropped and then was maintained at a constant level throughout the remainder of the incubation period. The further addition of ~-carnitine resulted in net synthesis of L-acetylcarnitine before reaching a steady state. Both pyruvate and lactate maintained maximal acetylation of ccarnitine. The most significant inhibition of L-acetylcarnitine oxidation was seen with ~-carnitine, although L-palmitoylcarnitine was inhibitory. By inhibiting L-acetylcarnitine oxidation, Gcarnitine would help regulate its own acetylation state. Childress et al. (10) reported that L-carnitine inhibited the rate of L-acetylcarnitine oxidation, attributing this to the readily reversible nature of the carnitine acetyltransferase. Thus, it is possible that as L-carnitine is increased, free CoA increases and this would make less acetyl-CoA available for citrate synthetase. More recently, Danks and Chappell (19) have postulated that L-carnitine probably competes with r,-acetylcarnitine for a particular site on the mitochondrial
352
HUTSON,
VAN
membrane, not at the carnitine acetyltransferase. Either postulate, or both, are adequate explanations for the results obtained with sperm. Note, however, that carnitine does not affect L-palmitoylcarnitine oxidation. L-Palmitoylcarnitine supported a slow rate of respiration in filipin-treated sperm. In brown adipose tissue mitochondria oxidizing palmitoylcarnitine, the decreased respiratory rates were correlated with dehydration of the mitochondrial matrix space associated with increasing concentrations of sucrose in the medium (27). We cannot rule out this possibility at present, but it is not a satisfactory explanation for the slow rate of oxidation seen in hypotonitally treated rabbit sperm in KC1 medium (14). The data also show that cpalmitoylcarnitine was a weak inhibitor of pyruvate oxidation. Bremer (9) reported that L-acetylcarnitine did not interfere with the oxidation of r,-palmitoylcarnitine in heart mitochondria; this agrees with the data in bovine sperm. Although endogenous fatty acid oxidation could be important during epididymal transit (quiescent state) (131, carbohydrate metabolism predominates after ejaculation when the energy demand is high. Bovine epididymal spermatozoa seem to be uniquely well suited for oxidizing both the aerobic and anaerobic end products of carbohydrate metabolism. In an anaerobic environment, energy is supplied by glycolysis and lactate accumulates. Depending on the available substrate, in an aerobic environment, energy is supplied by glycolysis and/or lactate or pyruvate oxidation. In the female genital tract, oxygen tensions are sufficiently high to support an aerobic type of metabolism (28). In in vitro systems, both glucose (mouse) (29) and pyruvate (guinea pig) (30) have been implicated as the major energy source during sperm capacitation. REFERENCES 1. CLAUSEN, J. (1969) Biochem. J. 111, 207-218. 2. MACHALXI DE DOMENECH, E., DOMENECH, C. E., AOKI, A., AND BLANCO, A. (1972)Biol. Reprod. 6, 136-147. 3. BLANCO, A., ZINKHAM, W. H., AND WALKER, D. G. (1975) in Isozymes (Markert, C. L., ed.),
DOP,
AND
4. 5. 6. 7.
a. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
19. 20.
21. 22. 23. 24. 25. 26. 27. 28.
29. 30.
LARDY
Vol. 3, pp. 297-312, Academic Press, New York. VAN DOP, C., HUTSON, S. M., AND LARDY, H. A. (1976) J. Biol. Chem. 252, 1303-1308. HUTSON, S. M., VAN DOP, C., AND LARDY, H. A. (1976) J. Biol. C&m. 252, 1309-1315. CASILLAS, E. R., AND ERICKSON, B. J. (1975) Biol. Reprod. 12, 275-283. MILKOWSKI, A. L., BABCOCK, D. F., AND LARDY, H. A. (1976) Arch. Biochem. Biophys. 1’76, 250-256. BREMER, J. (1962) J. Biol. Chem. 237,2228-2231. BREMER, J. (1965) B&him. Biophys. Actu 104, 581-590. CHILDRESS, C. C., SACKTOR, B., AND TRAYNOR, D. R. (1967) J. Biol. Chem. 242, 754-760. CASUAS, E. R. (1972) Biochim. Biophys. Acta 280, 545-551. HAMILTON, D. W., AND ORSON, G. E. (1976) J. Reprod. Fert. 46, 195-202. LARDY, H. A., AND PHILLIPS, P. H. (1941)Amer. J. Physiol. 134, 542-548. KEYHANI, E., AND STOREY, B. T. (1973) Fert. Steril. 24, 864-871. MORTON, B. E., AND LARDY, H. A. (1967) Biochemistry 6, 57-61. LANOUE, K., NICKLAS, W. 6., AND WILLIAMSON, J. R. (1970) J. Biol. Chem. 245, 102-111. PANDE, S. V., AND BLANCHAER, M. C. (1971) J. Biol. Chem. 246, 402-411. SHUG, A., LERNER, E., ELSON, C., AND SHRAGO, E. (1971) Biochem. Biophys. Res. Commun. 43, 557-563. DANKS, S. M., AND CHAPPELL, J. B. (1975)FEBS Lett. 59, 230-233. STOREY, B. T., AND KAYNE, F. J. (1976) in Ninth Annual Meeting of the Society for the Study of Reproduction, Abstract No. 61. BROOKS, D. E., AND MANN, T. (1972) Biochem. J. 129, 1023-1034. MILKOWSKI, A. L., AND LARDY, H. A., (1977) Arch. Biochem. Biophys. 181, 270-277.. LANOUE, K. F., BRYLA, J., AND WILLIAMSON, J. R. (1972) J. Biol. Chem. 247, 667-679. HANSFORD, R. G., AND JOHNSON, R. N. (1975) J. Biol. Chem. 250, 8361-8375. EVERSE, J., AND KAPLAN, N. 0. (1973) Aduan. Enzymol. 37, 61-133. ZINKHAM, W. H., BLANCO, A., AND CLOWRY, L. (1964)Ann. N.Y. Acad. Sci. 121, 571-588. NICHOLLS, D. G., GRAW, H. J., AND LINDBERG, 0. (1972) Eur. J. B&hem. 31, 526-533. BISHOP, D. W. (1956) in Proceedings of the Third International Congress on Animal Reproduction Physiology, p. 53, Brown Knight and Truscott, London. HOPPE, P. C. (1976) Biol. Reprod. 15, 39-45. ROGERS, B. J., AND YANAGIMACHI, R. (1975) Biol. Reprod. 13, 568-575.
OF
BIOCHEMISTRY
Metabolism
AND
BIOPHYSICS
of Pyruvate
for Enzyme
Research
and
345-352
(1977)
and Carnitine Esters in Bovine Sperm Mitochond rial
S. M. HUTSON, Institute
181,
C. VAN DOP,2
AND
the Department of Biochemistry, Wisconsin 53706 Received
December
Epididymal
H. A. LARDY University
of Wisconsin,
Madison,
6, 1976
Filipin-treated bovine epididymal spermatozoa have been used to study mitochondrial L-acetylcarnitine, t-palmitoylcarnitine, and pyruvate metabolism. The cells were supplemented with malatc to allow rapid rates of substrate oxidation. The rate of Lpahnitoylcamitine-supported state 3 respiration was slow. In contrast, pyruvate, acetylcarnitine, or lactate supported rapid and approximately equal respiratory rates. LPalmitoylcarnitine was a weak inhibitor of pyruvate-supported respiration and pyruvate use and a more potent inhibitor of L-acetylcarnitine. L-Camitine was an effective inhibitor of L-acetylcamitine oxidation; however, it did not influence L-palmitoylcarnitine oxidation or inhibit pyruvate utilization. Pyruvate (1.4 mM) disappearance was rapid and was complete within 6-7 min; the lactate produced during pyruvate metabolism was then oxidized. ATP synthesis was constant throughout the 20-min incubation. With pyruvate plus L-acetylcamitine as substrate, the L-acetylcarnitine concentration initially dropped and then recovered to a level that was dependent on free camitine addition. Data obtained from experiments using [2-14C]pyruvate indicated that the 14C label from pyruvate and lactate entered the L-acetylcamitine pool and labeling was maximal when free n-camitine was added. The rate of citrate synthesis was maximal when pyruvate was being metabolized; the largest total accumulation occurred when all three substrates were included in the incubation. The data suggest that the high NAD+/ NADH maintained during pyruvate metabolism may restrict flux through the citric acid cycle. The relationships of L-camitine and the L-camitine esters to pyruvate metabolism are discussed.
In mammalian sperm, the intramitochondrial location of lactate dehydrogenase-X (l-3) allows pyruvate to oxidize the NADH produced from its own metabolism (4, 5). Metabolism of pyruvate in these cells is exclusively intramitochondrial (4, 5). Recent reports (4,6,7) have also shown that bovine spermatozoa oxidizing pyruvate maintain high L-acetylcarnitine:carnitine ratios. The effects of L-carnitine and L-acetylcarnitine on pyruvate metabolism in heart and blowfly flight muscle mitochondria have been well documented (8-10). Casillas (11) has shown that in sonicated 1 Supported by Grants AM 10,334 and HD from the National Institutes of Health. 2 Recipient of Training Grant No. 2T05-GM from the National Institutes of Health.
spermatozoa, L-carnitine enhances the rate of palmitate oxidation. However, in ejaculated bovine spermatozoa, the addition of L-carnitine depressed fatty acid oxidation and stimulated incorporation of palmitate into 1,kdiglycerides (12). Bovine epididymal spermatozoa can oxidize endogenous lipids (13), but, in hypotonically treated rabbit epididymal spermatozoa, Lpalmitoylcarnitine oxidation was slow (14). Recently, we have used the polyene antibiotic, filipin, to study mitochondrial metabolism in bovine epididymal spermatozoa (5). Filipin renders the spermatozoan cell membrane permeable to small molecules (15). L-Carnitine addition to filipintreated spermatozoa metabolizing pyruvate resulted in a linear rate of cacetylcarnitine synthesis, which was maximal
08630 01932 345
Copyright All rights
0 1977 hy Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
346
HUTSON,
VAN DOP, AND LARDY
when oxalacetate was limiting (no added malate). The purpose of the present study was to examine the relationship(s) between metabolism of L-acetylcarnitine, long-chain acylcarnitine, and pyruvate. In addition, the effects of L-carnitine on the metabolism of these substrates have been examined. MATERIALS AND METHODS Preparation and treatment of bovine spermatozoa. Preparation, filipin treatment, and incubation of the bovine epididymal spermatozoa were as previously described (5). The standard incubation medium contained 15 mM Tris-phosphate, 10 mM MgSO,, 0.5 mM Tris-ADP, 35 mM 2-deoxyglucose, 0.1 mglml of hexokinase (dialyzed vs 15 mM Trisphosphate, pH 7.4, immediately before use), 250 mM mannitol, and 15 mM Tris(Cl), pH 7.4. When added, carnitine (pH 7.4, Tris) was 50 mM (5). All experiments were conducted at 37°C. Measurement of respiratory rates. Respiratory rates were measured using a Clark electrode attached to a Gilson oxygraph (Gilson Medical Electronics, Middleton, Wisconsin (5)). Radioactive experiments. Conversion of pyruvate to other metabolites was followed by incubating sperm suspensions (volume, 1.0 ml) in stoppered scintillation vials with 1.4 mM pyruvate containing 0.6 to 1.0 &i of [2X!lpyruvate. The concentrations of all stock substrate solutions were measured ensymatically. “C-labeled metabolites were separated by the chromatographic procedure of LaNoue et al. (16). Details of the incubation procedure and chro-
matographic separation pattern for tilipin-treated bovine epididymal sperm have been described (51. Assay methods. Pyruvate, lactate, malate, free Lcarnitine, L-acetylcarnitine, long-chain acylcarnitine, 2-deoxyglucose g-phosphate, glutamate, citrate, aspartate, /3-hydroxybutyrate, and fumarate were measured using standard techniques (4, 5). Materials. The sources of bovine epididymides, enzymes, L-carnitine, filipin, substrates, and [2‘Qpyruvate were as previously described (4, 5). LAcetylcarnitine and L-palmitoylcarnitine were obtained from P-L Biochemicals. RESULTS
Effects of the Various Substrates on Sperm Respiration
Malate (0.8 mM) did not support a significant rate of respiration but was included in all of the incubations because it was necessary for significant rates of palmitoylcarnitine or acetylcarnitine oxidation. cPalmitoylcarnitine-supported state 3 (ADP present) respiration was slow (Fig. 1A). Concentrations of L-pahnitoylcarnitine of from 12.5 to 200 PM supported maximal rates of respiration; at 400-500 PM, the surfactant effect was significant and respiration was inhibited. Unlike palmitoyl-CoA, palmitoylcarnitine is not a potent inhibitor of the adenine nucleotide translocase (17, 18). Sperm respiration supported by pyruvate or L-acetylcarnitine was inhibited by
FIG. 1. Effects of the various substrates on sperm respiration. Sperm were incubated in the standard incubation medium as described under Materials and Methods. The sperm concentrations were 2.8 x lOa (A-C) and 1.8 x 10s (D) cells/ml. Pyruvate and L-acetylcarnitine were 1.30 and 1.75 mxr, respectively. Malate (0.77 mM) was included in all incubations. The numbers in parentheses are the respiratory rates expressed as nanogram atoms of oxygen per 10s cells per minute.
METABOLISM
OF SPERM MITOCHONDBIA
increasing concentrations of L-palmitoylcarnitine (Figs. 1B and 10 With pyruvate as the substrate, 100 PM palmitoylcarnitine (the concentration used in subsequent metabolite experiments) inhibited respiration about 20%. L-Carnitine completely reversed the inhibitory effect of Lpalmitoylcarnitine on pyruvate oxidation (data not shown). With L-acetylcarnitine as substrate, 100 PM L-palmitoylcarnitine inhibited respiration significantly (Fig. 10. In contrast to the case with pyruvate (51, respiration supported by L-acetylcarnitine was progressively depressed by increasing concentrations of L-carnitine (Fig. 1D). Maximal inhibition was 75%. rEarmtine inhibits ~acetylcarnitine oxidation in blowfly flight muscle mitochondria (10, 19). In hypotonically treated rabbit spermatozoa, the L-acetylcarnitine-supported respiratory rate was about 25% of that of pyruvate, and L-acetylcarnitine inhibited the rate of pyruvate oxidation (14). In filipin-treated bovine epididymal sperm, Lacetylcarnitine was oxidized as well as pyruvate and had no effect on the pyruvatesupported respiratory rate. In other experiments (data not shown), lactate supported a respiratory rate equal to that with pyruvate. In combination with pyruvate, the respiratory rate was about the same as with either substrate alone. L-Acetylcarnitine and Pyruvate Metabolism The state 3 rate (ADP present) of Lacetylcarnitine-malate metabolism in filipin-treated spermatozoa is shown in Fig. 2A. GAcetylcarnitine disappearance was always faster during the first 4 min than during the remainder of the incubation [0.042 -+ 0.017 (4) and 0.130 f 0.002 (3) pmol used/loH cells at 4 and 20 min, respectively]. Malate disappearance always paralleled citrate synthesis and is usually not included in subsequent figures. As shown in Fig. 2B, pyruvate addition to cells metabolizing L-acetylcarnitine largely blocked Gacetylcarnitine disappearance. After an initial drop, n-acetylcarnitine remained at 85% of its initial
347
FIG. 2. Comparison of sperm mitochondrial Lacetylcarnitine and pyruvate metabolism. Twicewashed iilipin-treated cells were incubated in the standard incubation medium (see Materials and Methods). The sperm concentration was 9.9 x 10’ cells/ml. Substrate additions were as follows: PyNvate, 1.64 mM; cacetylcarnitine, 2.32 m&r; malate, 0.77 mM. The symbols used are: L-acetylcarnitine (a); malate (0); citrate (A); pyruvate (W); lactate (0).
concentration throughout the rest of the 20-min incubation. At the pyruvate and cell concentrations used in these experiments (1.4 mM and 6-10 x 10s cells/ml, respectively), the rate of pyruvate disappearance was linear and complete within 6-7 min after pyruvate addition (see Fig. 3). In agreement with previous reports concerning heart and blowfly flight muscle mitochondria (9, lo), L-acetylcarnitine addition did not affect the rate of pyruvate use (compare Figs. 2B and 20. Lactate appeared to be metabolized in preference to L-acetylcarnitine after all of the pyruvate had been used (Figs. 1B and 10. It seems likely that lactate is being oxidized via lactate dehydrogenase-X and pyruvate dehydrogenase; some of the acetate is shunted into acetylcarnitine and the rest is being oxidized via the citric acid cycle. In Figs. 2B and 2@, at 4 min, citrate accumulation was already 50% of the final value at 20 min; thus, citrate synthesis was maximal when pyruvate was being metabolized. The total amount of citrate that accumulated was largest in the presence af both substrates (Fig. 2B). Effects of L-Palmitoylcarnitine L-Palmitoylcarnitine (0.17 mM) inhibits the decarboxylation of pyruvate in rat
348
HUTSON,
VAN
heart mitochondria (9). L-Palmitoylcarnitine (100 PM) was a very weak inhibitor of pyruvate metabolism (Figs. 3A and 3B); in other experiments, maximal inhibition of the rate of pyruvate use was never greater than 20%. As in rat heart (9), pyruvate significantly inhibited L-palmitoylcarnitine disappearance (compare Figs. 3B and 3C). Pyruvate inhibition of L-palmitoylcarnitine metabolism was not further characterized, because the long-chain acylcarnitine extraction procedure was unreliable. The effects of L-palmitoylcarnitine on Lacetylcarnitine and pyruvate metabolism are illustrated in Figs. 4A-4C. The corresponding controls are shown in Fig. 2. LPalmitoylcarnitine inhibited cacetylcarnitine disappearance by about 35% (Fig. 4A); in other experiments, the inhibition ranged from 35-55%. Direct comparison of the data obtained in the respiration and metabolism experiments is not possible, because cell concentrations were two to four times greater in the experiments in which metabolites were measured. Citrate synthesis was increased by palmitoylcarnitine and was maximal with all three substrates present (Fig. 40 When all three substrates were included in the incubation, L-acetylcarnitine initially decreased was then reacetylated to 94% of its original concentration (Fig. 40.
FIG. in Fig. malate lactate
DOP,
AND
Effects
LARDY
of L-Carnitine
rK!arnitine has a significant function in carbohydrate metabolism in bovine epididymal sperm (4, 6, 7) and filipin-treated sperm (5). In the filipin-treated sperm, Lcarnitine addition to cells metabolizing Lacetylcarnitine inhibited acetylcarnitine use about 50%, with a concomitant decrease in malate use and citrate synthesis (Fig. 5A). In contrast to the data obtained with acetylcarnitine, carnitine did not influence L-palmitoylcarnitine oxidation (data not shown). The corresponding controls without L-carnitine are shown in Fig. 2. As shown previously, L-carnitine did not alter the rate of pyruvate use, but consistently decreased the amount of pyruvate that was converted to lactate by 5-10%; part of the pyruvate was preferentially shunted into L-acetylcarnitine (Table I and Ref. 5). The data in Figs. 5B and 5C suggest that lactate must have been a source of acetyl-CoA for carnitine acetyltransferase. rCarnitine addition also reduced citrate accumulation (compare Figs. 2 and 5). In other experiments (data not shown), Lcarnitine eliminated any effects of ~-palmitoylcarnitine on pyruvate use. Metabolism
The
of Malate and [2-WPyruvate
metabolism
of malate
and
3. Effect of L-palmitoylcarnitine on pyruvate metabolism. Conditions were the same as 1. The sperm concentration was 1.1 x lo@ cells/ml. F’yruvate was added at 1.36 mM, at 0.77 mM, and L-palmitoylcarnitine at 100 pM. The symbols used are: pyruvate (a); (0); net free carnitine (A).
[2-
METABOLISM
OF SPERM
FIG. 4. Metabolism of L-acetylcamitine, L-palmitoylcamitine, and pyruvate. Incubation conditions and cell and substrate concentrations were the same as in Fig. 2; the symbols are the same as in Fig. 2. In addition, L-palmitoylcarnitine was 100 PM.
MITOCHONDRIA
349
In general, label did not pass through the citric acid cycle more than once. Similarly, the malate pool was not replenished with malate containing 14C. Even though greater than 80% of the malate had been used by 20 min, the final specific activity of the remaining malate ranged from 20 to 30% of the initial pyruvate. Total fumarate and aspartate were less than 10 nmol/108 cells at any time point. Glutamate was about 10 nmol/lOs cells and did not vary under any conditions tested. Free acetoacetate and /3-hydroxybutyrate were not detectable. Oxidative Phosphorylation
The synthesis of 2-deoxyglucose B-phos14C]pyruvate is summarized in Tables I phate was used as a measure of ATP production (15), but remains a qualitative and II. The release of ‘4c02 was low and measure because of ATPase activity (5). even after 20 min was less than 20% of the ATP synthesis in cells oxidizing acetylcartotal pyruvate added. As in blowfly flight the incubamuscle mitochondria (3), L-carnitine ap- nitine was linear throughout tion period (Fig. 6-1) and the average of peared to decrease 14C02 release; but the percentage was quite variable. Less 14C four values at 20 min was 0.653 + 0.064 ~mol/lO* cells. Although L-palmitoylcarnilabel accumulated in COz and succinate tine inhibited acetylcarnitine disappearwhen unlabeled L-acetylcarnitine and Lance, this was not reflected in the rate of palmitoylcarnitine were added. At 20 min, oxidative phosphorylation (Fig. 6-2). Genthe ‘4c label had accumulated in most citerally, the effects of the various comric acid cycle intermediates. However, pounds on the rate of oxidative phosphomost of the 14C label was concentrated in rylation supported by acetylcarnitine relactate, citrate, and, with L-carnitine, in Lflected the results seen in the respiration acetylcarnitine as well. and metabolism experiments. In cells oxiThe data indicate that label from lactate entered the L-acetylcarnitine pool (Table I, Fig. 2), for pyruvate had disappeared during the first 6-7 min and acetylcarnitine continued to accumulate ‘4c label. With all three substrates plus carnitine, there was net acetylcarnitine synthesis and the largest 14C labeling. The fractions containing L-acetylcarnitine were analyzed after alkaline hydrolysis (Table II). If a source of carnitine was available, acetate comprised 8595% of the labeled material; without carnitine, acetate comprised about 45%. An unknown component, tentatively identified as lactate or /3-hydroxyFIG. 5. Effects of L-carnitine on L-acetylcarnitine butyrate (8, ranged from 2-5 nmol/108 and pyruvate metabolism. Conditions were the cells, while the unhydrolyzed compound same as in Fig. 2; the symbols are the same as in (probably alanine) (16) was constant at Fig. 2. The sperm concentration was 7.8 x lo* cells/ about 1.5 nmol. ml. L-Camitine was 50 mM, malate was 0.96 mM The specific activity of the [14C]citrate and, when present, pyruvate was 1.51 mM. L-Acetylwas similar to that of the [2-14C]pyruvate. camitine was 2.05 mM.
350
HUTSON,
VAN DOP, AND LABDY TABLE
EFFECT
I
OF L-CARNITINE, GACET~LCARNITINE, AND L-PALMITOYLCARNITINE ON THE 14C! LABELING METABOLITES IN SPERM OXIDIZING MALATE AND [2-14C]Py~w~~~a
Other additions
None L-Camitine L-Acetylcamitine + L-palmitoylcamitine L-Acetylcarnitine + L-palmitoylcamitine + L-camitine
OF
Sampp;t (min)
Pyruvate metabolizedb
I Acetylcamitine”
II Acetate glutamate6
III Lactat&
VIII Citrate6
v succinate”
4 20 20 4 20 20
135 189 157 81 161 249
9 9 10 11 52 42 (203)”
3.5 14 17 4 12 32
74 50 55 39 33 99
25 50 35 26 30 60
9 10 8 3
3 19 9 12 2
7 38 21 2 19 10
20
236
79 (325)
32
41
82
2
3
6
IbEate*
14coI*
a Conditions are the same as in Fig. 1. The sperm concentration ranged from 6.2 to 8.1 x lOa cells/ml. Pyruvate ranged from 1.1 to 1.44 mM and malate was 0.8 mM. Initial r.-acetylcamitine was 1.72 mM (275 nmol/lO* cells) and L-palmitoylcamitine was 100 pM. At 20 min, 99% of the initial pyruvate had been used. Roman numerals heading six of the columns designate the chromatographic fraction and the major component(s) of the fraction. * Nanomoles per lo* cells, based on the specific activity of the [2-“Clpyruvate. c The enzymatically determined concentration of acetyl camitine is given in parentheses. TABLE ANALYSIS
II
OF THE ALKALINE COMPONENT
Additions
HYDRQLYSATE
Ia Unhydrolyzedb 1.8 1.3 1.0
Acet&e*
OF
Unknown”
None 4.1 3.9 L-Camitine 50.2 1.2 n-Acetylcarnitine 35.4 5.4 + L-palmitoylcarnitine 1.3 76.1 2.0 L-Acetylcarnitine + L-palmitoylcamitine + L-carnitine n The pooled fractions from Peak I (see Table I) were rechromatographed on Dowex I after alkaline hydrolysis. b Nanomoles per lo8 cells per 20 min; calculations are based on the specific activity of the [2“Clpyruvate.
dizing palmitoylcarnitine, ATP synthesis was slow and was not influenced by carnitine (Fig. 6-4). However, carnitine was inhibitory in cells oxidizing acetylcarnitine (Fig. 6-5); further addition of cpalmitoylcarnitine resulted in an intermediate rate of synthesis (Fig. 6-3). Thus, while acetylcarnitine oxidation was inhibited by carnitine, palmitoylcarnitine oxidation was unaffected. The rate of 2-deoxyglucose 6-phosphate
synthesis in cells metabolizing pyruvate was constant for 15 to 20 min (Fig. 6-6). G Carnitine, L-acetylcarnitine (Fig. 6-71, or L-palmitoylcarnitine, or combinations of these substrates, did not have any effect on this rate. The typical variation within a single experiment was 2-8%; among different cell populations, the variation was approximately 15%. DISCUSSION
The data presented here confrm earlier observations made on intact cells (4) which suggested that lactate was not metabolized when pyruvate was present. However, after all of the pyruvate was used, lactate was metabolized. Lactate use was much slower than pyruvate use, but lactate-supported respiration was equal to that with pyruvate. Storey and Kayne (20) have reported that, in hypotonically treated rabbit epididymal sperm, lactate was oxidized more rapidly than pyruvate. This difference may be the result of the differing treatments or species. ~Acetylcarnitine or lactate plus malate gave comparable reduction of mitochondrial pyridine nucleotides (20). Spermatozoa metabolizing pyruvate maintain a high NAD+/NADH (21, 22). Data presented here and earlier work
METABOLISM
0
5
I5
OF SPERM
20
FIG. 6. Effects of the various treatments on 2deoxyglucose 6-phosphate synthesis. Incubation conditions and substrate and cell concentrations were the same as in Figs. 2 and 4. Malate was included in all incubations, Cells were metabolizing the following substrates: L-acetylcarnitine (11, Lacetylcarnitine plus L-palmitoylcarnitine (21, both substrates plus L-carnitine (31, L-palmitoylcarnitine plus L-carnitine (41, n-acetylcamitine plus L-carnitine (51, pyruvate (61, and pyruvate plus L-acetylcarnitine (7).
(5) suggested that in filipin-treated cells pyruvate is incompletely oxidized. During pyruvate metabolism, citrate accumulation was large and 14C02 release was low. Furthermore, the data imply that lactate, and probably tacetylcarnitine, are completely oxidized via the citric acid cycle. This leads directly to the possibility that when the mitochondrial pyridine nucleotides are highly oxidized, flux through the citric acid cycle is limiting for pyruvate oxidation. The block may be at isocitrate is supdeh ydrogenase . This postulate ported by the pattern of citrate accumulation. Control of flux through the citric acid cycle in sperm may be very different from that seen in heart mitochondria (16, 23, 24).
Ever-se and Kaplan (25) proposed that under normal in uiuo conditions, the Htype lactate dehydrogenase is prevented from reducing pyruvate to lactate, while maintaining its full capacity to oxidize lactate. A high NAD+/NADH and a high pyruvate/lactate would favor abortive complex formation; lowering the pyruvate/lac-
MITOCHONDRIA
351
tate would favor oxidation of lactate. Certain kinetic properties of lactate dehydrogenase-X, in particular the striking inhibition by pyruvate, have led Blanco et al. (3) to extend this hypothesis to the sperm enzyme. Lactate dehydrogenase-X represents 80% or more of the total lactate dehydrogenase activity of spermatozoa (26). In sperm mitochondria, pyruvate was preferentially reduced to lactate. As shown previously (41, pyruvate reduction occurred over a wide range of pyruvate/lactate even with a high NAD+/NADH. Thus, the mitochondrial NAD+/NADH ratio may be the most important regulatory parameter for lactate dehydrogenase-X. Casillas and Erickson (6) postulated that, in spermatozoa, the acetylcarnitine:carnitine transferase system is used to buffer against rapid changes in the concentration of acetyl-CoA. More recently, Milkowski et al. (7) and Van Dop et al. (4) have suggested that L-acetylcarnitine may also serve as a source of “active” acetate. Childress et al. (10) postulated a similar role for acetylcarnitine in insect flight muscle (under oxalacetate-limiting conditions). With pyruvate and L-acetylcarnitine as the substrates, the L-acetylcarnitine concentration initially dropped and then was maintained at a constant level throughout the remainder of the incubation period. The further addition of ~-carnitine resulted in net synthesis of L-acetylcarnitine before reaching a steady state. Both pyruvate and lactate maintained maximal acetylation of ccarnitine. The most significant inhibition of L-acetylcarnitine oxidation was seen with ~-carnitine, although L-palmitoylcarnitine was inhibitory. By inhibiting L-acetylcarnitine oxidation, Gcarnitine would help regulate its own acetylation state. Childress et al. (10) reported that L-carnitine inhibited the rate of L-acetylcarnitine oxidation, attributing this to the readily reversible nature of the carnitine acetyltransferase. Thus, it is possible that as L-carnitine is increased, free CoA increases and this would make less acetyl-CoA available for citrate synthetase. More recently, Danks and Chappell (19) have postulated that L-carnitine probably competes with r,-acetylcarnitine for a particular site on the mitochondrial
352
HUTSON,
VAN
membrane, not at the carnitine acetyltransferase. Either postulate, or both, are adequate explanations for the results obtained with sperm. Note, however, that carnitine does not affect L-palmitoylcarnitine oxidation. L-Palmitoylcarnitine supported a slow rate of respiration in filipin-treated sperm. In brown adipose tissue mitochondria oxidizing palmitoylcarnitine, the decreased respiratory rates were correlated with dehydration of the mitochondrial matrix space associated with increasing concentrations of sucrose in the medium (27). We cannot rule out this possibility at present, but it is not a satisfactory explanation for the slow rate of oxidation seen in hypotonitally treated rabbit sperm in KC1 medium (14). The data also show that cpalmitoylcarnitine was a weak inhibitor of pyruvate oxidation. Bremer (9) reported that L-acetylcarnitine did not interfere with the oxidation of r,-palmitoylcarnitine in heart mitochondria; this agrees with the data in bovine sperm. Although endogenous fatty acid oxidation could be important during epididymal transit (quiescent state) (131, carbohydrate metabolism predominates after ejaculation when the energy demand is high. Bovine epididymal spermatozoa seem to be uniquely well suited for oxidizing both the aerobic and anaerobic end products of carbohydrate metabolism. In an anaerobic environment, energy is supplied by glycolysis and lactate accumulates. Depending on the available substrate, in an aerobic environment, energy is supplied by glycolysis and/or lactate or pyruvate oxidation. In the female genital tract, oxygen tensions are sufficiently high to support an aerobic type of metabolism (28). In in vitro systems, both glucose (mouse) (29) and pyruvate (guinea pig) (30) have been implicated as the major energy source during sperm capacitation. REFERENCES 1. CLAUSEN, J. (1969) Biochem. J. 111, 207-218. 2. MACHALXI DE DOMENECH, E., DOMENECH, C. E., AOKI, A., AND BLANCO, A. (1972)Biol. Reprod. 6, 136-147. 3. BLANCO, A., ZINKHAM, W. H., AND WALKER, D. G. (1975) in Isozymes (Markert, C. L., ed.),
DOP,
AND
4. 5. 6. 7.
a. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
19. 20.
21. 22. 23. 24. 25. 26. 27. 28.
29. 30.
LARDY
Vol. 3, pp. 297-312, Academic Press, New York. VAN DOP, C., HUTSON, S. M., AND LARDY, H. A. (1976) J. Biol. Chem. 252, 1303-1308. HUTSON, S. M., VAN DOP, C., AND LARDY, H. A. (1976) J. Biol. C&m. 252, 1309-1315. CASILLAS, E. R., AND ERICKSON, B. J. (1975) Biol. Reprod. 12, 275-283. MILKOWSKI, A. L., BABCOCK, D. F., AND LARDY, H. A. (1976) Arch. Biochem. Biophys. 1’76, 250-256. BREMER, J. (1962) J. Biol. Chem. 237,2228-2231. BREMER, J. (1965) B&him. Biophys. Actu 104, 581-590. CHILDRESS, C. C., SACKTOR, B., AND TRAYNOR, D. R. (1967) J. Biol. Chem. 242, 754-760. CASUAS, E. R. (1972) Biochim. Biophys. Acta 280, 545-551. HAMILTON, D. W., AND ORSON, G. E. (1976) J. Reprod. Fert. 46, 195-202. LARDY, H. A., AND PHILLIPS, P. H. (1941)Amer. J. Physiol. 134, 542-548. KEYHANI, E., AND STOREY, B. T. (1973) Fert. Steril. 24, 864-871. MORTON, B. E., AND LARDY, H. A. (1967) Biochemistry 6, 57-61. LANOUE, K., NICKLAS, W. 6., AND WILLIAMSON, J. R. (1970) J. Biol. Chem. 245, 102-111. PANDE, S. V., AND BLANCHAER, M. C. (1971) J. Biol. Chem. 246, 402-411. SHUG, A., LERNER, E., ELSON, C., AND SHRAGO, E. (1971) Biochem. Biophys. Res. Commun. 43, 557-563. DANKS, S. M., AND CHAPPELL, J. B. (1975)FEBS Lett. 59, 230-233. STOREY, B. T., AND KAYNE, F. J. (1976) in Ninth Annual Meeting of the Society for the Study of Reproduction, Abstract No. 61. BROOKS, D. E., AND MANN, T. (1972) Biochem. J. 129, 1023-1034. MILKOWSKI, A. L., AND LARDY, H. A., (1977) Arch. Biochem. Biophys. 181, 270-277.. LANOUE, K. F., BRYLA, J., AND WILLIAMSON, J. R. (1972) J. Biol. Chem. 247, 667-679. HANSFORD, R. G., AND JOHNSON, R. N. (1975) J. Biol. Chem. 250, 8361-8375. EVERSE, J., AND KAPLAN, N. 0. (1973) Aduan. Enzymol. 37, 61-133. ZINKHAM, W. H., BLANCO, A., AND CLOWRY, L. (1964)Ann. N.Y. Acad. Sci. 121, 571-588. NICHOLLS, D. G., GRAW, H. J., AND LINDBERG, 0. (1972) Eur. J. B&hem. 31, 526-533. BISHOP, D. W. (1956) in Proceedings of the Third International Congress on Animal Reproduction Physiology, p. 53, Brown Knight and Truscott, London. HOPPE, P. C. (1976) Biol. Reprod. 15, 39-45. ROGERS, B. J., AND YANAGIMACHI, R. (1975) Biol. Reprod. 13, 568-575.