Recovery metabolism role of mitochondria CHRISTOPHER Department
of trout white muscle:
D. MOYES, PATRICIA of Zoology, University of British
M. SCHULTE, Columbia,
Moyes, Christopher D., Patricia M. Schulte, and Peter W. Hochachka. Recovery metabolism of trout white muscle: role of mitochondria. Am. J. Physiol. 262 (Regulatory Integrative Comg. Physiol. 31): R295R304, 1992.-Recovery from burst exercise in fish is very slow. Lactate conversion to glycogenoccursprimarily within white muscleand must be fueled by mitochondrially produced ATP. In a parallel study we characterized the changes in tissue metabolites associatedwith burst exercise and recovery in rainbow trout (Oncorhynchus mykiss)white muscle.The present study examineswhether the mitochondrial capacity to produce ATP may limit the rate of recovery of trout white muscle.The cost (ATP l rnin-l *g-l) of glycogenresynthesis(0.05 pmol lactate converted* min-’ g tissue-l) was compared with the mitochondrial capacity to produce ATP. The cost of recovery can be met by only 3.5% of the maximal mitochondrial capacity. In fact, during recovery trout white musclemitochondria operate at a small fraction of their in vitro maximum. This capacity is suppressedin vivo by highly inhibitory ATP/ADP and limiting phosphate. The primary signal for increasedATP synthesisassociatedwith recovery is not a change in ATP/ADP but probably phosphate, elevated becauseof phosphocreatine hydrolysis and adenylate catabolism in the purine nucleotide cycle. At low ADP availability and suboptimal phosphate (6 mM), acidosisenhancesrespiration. At high respiratory rates mitochondrial pyruvate oxidation is sensitive to pyruvate concentration over the physiological range (apparent Michaelis constant = 35-40 PM). This sensitivity is lost at the low rates that approximate in vivo respiration. Changesin lactate do not affect the kinetics of pyruvate oxidation. Fatty acid oxidation may spare pyruvate and lactate for usein glyconeogenesis,primarily through allosteric inhibition of pyruvate dehydrogenaserather than covalent modification. l
AND
Vancouver,
PETER British
W. HOCHACHKA
Columbia
V6T 2A9, Canada
flounder recovery requires 8-12 h or more (19). Also, a number of indirect studies suggest that white muscle lactate is metabolized primarily in the white muscle. Injections of [“Cllactate into the blood remain primarily as lactate [skipjack tuna (39), flounder and salmon (19)]. Lactate turnover cannot account for the changes observed in white muscle lactate [salmon and flounder (19)], suggesting the blood and white muscle do not equilibrate. Studies using the glucose analogue deoxyglucose suggest that 40% of the glycogen resynthesized in white muscle after burst exercise in rainbow trout can be attributed to exogenous glucose uptake (T. G. West, P. M. Schulte, and P. W. Hochachka, unpublished observation). Furthermore, hepatocyte studies suggest that fish liver has insufficient capacity for gluconeogenesis from lactate to account for the rate of lactate disappearance (38). Because there is a net conservation of white muscle carbohydrate (glucosyl units + 2 X lactate) during exercise and recovery (32a), it is clear that oxidation is not an important route of lactate metabolism postexercise. The route for white muscle glycogen resynthesis from lactate is unclear. White muscle lacks adequate activities of pruvate carboxylase, one of the enzymes required for gluconeogenesis in liver (28). Alternate routes in mammals could involve malic enzyme plus phospho(enol)pyruvate carboxykinase (8) or reversal of pyruvate kinase (11). White muscle from most fish species lacks both pyruvate carboxylase and phospho(enoZ)pyruvate car-
boxykinase (e.g., Refs. 9,22), suggesting pyruvate kinase reversal may be the more important route of glyconeogenesis in fish. At present, direct evidence for any route ATP; ADP; phosphate; pyruvate; exercise; pH; purine nucleo- is lacking in fish. tide cycle Whatever the route of glycogen resynthesis, the ATP demands imposed by recovery metabolism (net glycogen HIGH-INTENSITY EXERCISE in mammalian white muscle is fueled by breakdown of glycogen, with formation of
lactate and accompanied by a tissue acidosis. Recovery metabolism involves reducing lactate concentration ([lactate]) to resting levels and repletion of glycogen stores. In mammals, if blood [lactate] is low, lactate is released and primarily oxidized in aerobic tissues, with a smaller proportion used as a glyconeogenic substrate (6). If perfusion [lactate] is high, conversion of lactate to glycogen within white muscle (glyconeogenesis) becomes increasingly important, especially in the more glycolytic fiber types (4, 29). Recovery from such exercise bouts typically takes several minutes to an hour (e.g., Ref. 15). Thus, in mammals, lactate metabolism postexercise is relatively fast and may involve several anabolic and catabolic tissues. Fish burst exercise and recovery differs from the mammalian pattern in a number of important aspects. The changes in metabolites are greater and more prolonged than in mammalian white muscle (19). Salmon and 0363-6119/92
$2.00 Copyright
synthesis) must be met by mitochondrial (aerobic) pathways. Comparison among the rates of recovery reported for various species [tuna > salmon > plaice (1, 21)]
reveals an apparent correlation with the mitochondrial density of the white muscle. Species with mitochondriarich white muscle recover faster than species with white muscle with low mitochondrial content. The present study investigates whether the ATP production by white muscle mitochondria limits the rate of glyconeogenesis. The cost of recovery is compared with mitochondrial capacity of the tissue. We also examine the influence of changes in ATP/ADP, pH, phosphate, and carbon metabolites on mitochondrial rate and substrate selection. MATERIALS AND METHODS Animals Rainbow trout (Oncorhynchus mykiss) of both sexes(400600 g) were held in continuously flowing fresh water at seasonal
temperatures. Animals were fed ad libitum four times a week.
0 1992 the American
Physiological
Society
R295
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R296
MITOCHONDRIAL
METABOLISM
OF TROUT
WHITE
MUSCLE
ExerciseProtocol
IsolatedMitochondrial Studies
The methodology involving exercise of fish is described in our parallel study (32a). Briefly, fish were exercisedto exhaustion in a swimtunnel and either killed immediately or allowed to recover for 2,4,8, or 24 h. An anesthetic (Somnotol) overdose technique was developed to kill the fish quickly with little struggling so asto minimize changesin labile metabolites.
Trout were killed by blow to the head. Approximately 40 g of white musclewascollected from an area extending from the dorsal fin to 3 cm anterior to the caudalpeduncle,including all tissue dorsal to the lateral line. After skinning the tissue,great care was taken to shear off any superficial red muscle.Tissue wasplaced on an ice-cold Petri dish, choppedwith razor blades, divided into three portions and addedto three Potter-Elvejhem homogenizers, each containing 30 ml isolation buffer. The composition of isolation buffer is as describedpreviously (23). Tissue was dispersedby two passesof a loosely fitting pestle followed by three passesof a more tightly fitting pestle. Homogenateswere combined and diluted to 500 ml with more isolation buffer and centrifuged 3 min at 2,660g. The supernatants were poured through four layers of cheeseclothand recentrifuged 5 min at 2,600 g. The supernatant was poured through eight layers of cheeseclothand centrifuged 10 min at 9,000g. Mitochondrial pellets were resuspendedin 10 ml isolation medium [minus bovine serum albumin (BSA)] and centrifuged 5 min at 8,000 g. The mitochondrial pellet was resuspended in l-2 ml BSA-free (to facilitate protein determinations) isolation medium and usedwithin 60 min.
Enzyme Activities Tissuehomogenateswere prepared in 9 vol of the following medium: 100 mM potassiumphosphate, 5 mM EDTA, 1 mM dichloroacetate, and 0.1% Triton X-100 at pH 7.0. Fluoride, often usedto inhibit pyruvate dehydrogenase-kinasewas not used as it inhibited pyruvate dehydrogenase(PDH) directly. Tissue was homogenizedby three bursts of an UltraTurrax grinder followed by three bursts of a Kontes sonicator. Homogenateswere centrifuged 10 min at 10,000g, with the resulting supernatantusedfor assaysof both phosphorylated active PDH (PDH,) and citrate synthase (CS). Activity of PDH, wasdetermined by measuringacetyl-CoAdependentacetylation of p-nitroaniline in the presenceof pigeonliver arylamine acetyltransferasepurified basedon Tabor et al. (36). Pigeon liver was frozen in liquid nitrogen and kept up to 2 mo before extraction. Tissue (100 g) was homogenized in 200 ml of 5 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-1 mM EDTA (pH 7.0). Homogenates were centrifuged 20 min at 12,006g. The pellets were rehomogenized in 200 ml of buffer and recentrifuged. The combined supernatantswere brought to 500 ml and addedto 400 ml icecold acetone. After centrifugation (10 min at 10,000g), the combinedsupernatantswere added to 1 liter acetone and centrifuged (5,000g for 10 min). The pellet wasdissolvedin 20 ml water for 60 min at 4°C and addedto 5 g alumina CT (pelleted from 50 ml suspension;Sigma Chemical). After 1 h of swirling (2 Hz), the suspensionwas centrifuged 7 min at 1,000g. The pellet waswashedtwice with 100 ml water before elution with 250 ml potassiumphosphatebuffer (100 mM, pH 7.7) in four aliquots. The enzyme wasconcentrated by Amicon Centriprep units and frozen (080°C) until needed.Each purification was checked for contaminating PDH, activity, and none was detected. PDH, activity in tissueextracts was assayedusing a PerkinElmer Lambda 2 spectrophotometer, interfaced with an IBM computer using PECSS (Perkin-Elmer) software to calculate enzyme activities. The l-ml assay mixture contained 1 mM MgCls, 0.5 mM EDTA, 0.5 mM cocarboxylase, 0.15 mM CoA, 2 mM NAD+, 5 mM /3-mercaptoethanol,0.1 mMp-nitroaniline, 200 mU a&mine acetyltransferase, 40 ~1 homogenate, and 100mM tris(hydroxymethyl)aminomethane (Tris; pH 7.7). Reaction was initiated by addition of 1 mM pyruvate and was linear for at least5 min. No activity wasdetectedin the absence of pyruvate. Activity was measuredwithin 30 min of homogenization. Attempts to fully activate fish musclePDH by purified pig heart PDH-phosphatasewere unsuccessful.To standardize for interindividual differences in mitochondrial density, PDH, activity is expressedagainst CS activity determined in the same homogenate. The CS assaycontained 0.3 mM acetyl-CoA, 0.2 mM 5,5’dithio-bis(2nitrobenzoic acid), and 100 mM Tris (pH 8.0) and was initiated by addition of 0.5 mM oxaloacetate. Blank rates (minus oxaloacetate)were ~5% of the CS rate. All enzyme assayson whole tissue extracts were performed at 25°C to obtain maximal sensitivity of the PDH, assay and minimize coupling enzyme requirements. Q10determinations were performed to allow conversion between tissue enzyme analysesand mitochondrial studiesperformed at l5OC.
Oxygen Consumption Determination of the rate of oxidation of physiological substrates was as describedpreviously (24) at pH 7.3 and 15OC. The state 3 rate is the rate of oxygen consumption in the presenceof ADP (0.3 mM). The state 4 rate is obtained after all the ADP hasbeen phosphorylated. The respiratory control ratio (RCR) equalsstate 3 divided by state 4. The oxygen consumption rate was also determined as a function of the ratio ATP/ADP. Assays were performed with 0.3-0.5 mg protein/ml assay medium. Injections of 7.5 mM ATP, 10 mM glucose,then increasing hexokinase levels were used to obtain an ATP/ADP closeto that seenin recovering fish (32a). Aliquots (500 ~1) were removed from parallel incubations and added to 500 ~1ethanol for deproteinization. The supernatants (10 min at 15,000 g) were frozen for ATP and ADP determinations by high-performance liquid chromatography (32a). The influence of MgC12was determined at subsaturating [ADP]. Mitochondria were addedto a cuvette containing assay medium (minus BSA), 1 mM pyruvate, 0.1 mM malate, and 150 U hexokinase with and without 5 mM MgClz. The high activity of hexokinase was usedto preclude an indirect effect of M$+ due to Mg2+-dependent stimulation of hexokinase. Reactions were started by addition of 5 or 2 PM ADP. The ADP levels were chosen to elicit submaximal rates that were dependent on ADP transport rate into the mitochondria, analogousto high ATP/ADP. The actual ATP/ADP wasnot determined. In experiments where we used a hexokinase ADP-regenerating system, ATP production rates were determined by assaying glucose6-phosphateplus fructose 6-phosphateproduction. Because such high activities of hexokinase were used, the relatively low contamination by phosphoglucoisomerase became significant. Approximately 10% of the glucose6=phosphate was converted to fructose 6-phosphate. Mitochondrial incubations (0.9 ml) were addedto 0.1 ml 70% perchloric acid and centrifuged 5 min at 15,060g. Aliquots of the supematant were neutralized by addition of 1 M Tris (40 mM final concn) and 10 M KOH. Glucose6-phosphateplus fructose g-phosphate was assayedin 50 mM Tris (pH 8.0) and 1 mM NADP+. The reaction was started by addition of 1 U glucose-6-phosphate dehydrogenaseplus 1 U phosphoglucoisomerase and was complete within 2 min.
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MITOCHONDRIAL
METABOLISM
OF TROUT
WHITE
Mitochondrial Pyruvate Oxidation
RESULTS
Pyruvate oxidation was assayedusing [1-14C]pyruvate. Becausethe radiolabeledcarbon is removed in the decarboxylation reaction of the PDH complex, “CO2 production representsthe flux through PDH in situ (within mitochondria). This technique was used to examine the influence of a number of potential effecters of mitochondrial metabolism. Also, some comparisonswere performed between mitochondria from trout white muscle and carp red muscle. Carp mitochondria were preparedaccordingto Moyes et al. (23) and assayedasdescribed here for the trout preparation. Small volumes of mitochondria were added to the assay medium used for oxygen consumption experiments to a total volume of 2 ml. Incubations were performed in 20-ml scintillation vials. A 5-min preincubation period was sufficient time to allow the vials to be cappedwith rubber stoppersfitted with plastic center wells. Reaction was started by injection of 100 ~1 of pyruvate (12C+ 14C,0.25 &i//rmol final sp act at each pyruvate concn) through the rubber cap, and vials were shaken (1 Hz) at 15OCfor 10 min unlessnoted otherwise. Incubations were terminated by a 200.~1injection of 70% perchloric acid. Hyamine hydroxide (150 ~1) was addedto the center well, and vials were shaken90 min at room temperature to collect 14C02. Filter paperswere addedto 10 ml of ACS-2 (Amersham), with 0.1% acetic acid to prevent background color formation and eliminate chemiluminescence.Sampleswere counted for 10min in a LKB 1214 RackBeta scintillation counter with programmedquench correction. All assayswere done in at least duplicate. A similar protocol was used to determine PDH, activities with solubilized mitochondria. The assay componentswere as describedfor tissue PDH, anaIysis except p-nitroaniline and a&mine acetyltransferasewere omitted and coenzyme A was elevated to 0.6 mM to prevent significant depletion in the longer incubations. Decarboxylation of [l-14C]pyruvate was measuredasbefore. The reaction was shown to be linear for at least 10 min using PDH that was inactivated by 20-min incubation of mitochondria with 50 PM palmitoylcarnitine. The linearity of the PDH, assay with inactivated PDH demonstrates that the assay procedure did not allow activation in vitro. Routinely, assay incubations were run for 6 min in at least duplicate. Studies examining the kinetics of pyruvate oxidation by intact mitochondria were performed under the conditions described above. Oxygen consumption rates provided estimates of pyruvate oxidation, which were usedto establishthe highest mitochondrial concentration that would not deplete pyruvate by X0% at the lowest [pyruvate] usedin the radiolabel studies. Preliminary studiesshowedthe rate of pyruvate oxidation was linear with respect to [protein] over the ranges used in this study and linear for at least 15 min. The influence of ADP availability was examined under two conditions. Experiments in state 4 (no added ADP) used high [protein] to obtain adequatedisintegrations-per-minute collection. State 3 experiments (+ADP) wereperformed with 0.3 mM ADP, which was not substantially depleted becausemitochondria were diluted and incubations were limited to 10 min. In someexperiments lactate (40 mM) was added5 min before addition of pyruvate.
Mitochondrial
Statistics Where statistical comparisonswere required, an analysis of variance was usedwith Tukey’s multiple comparisontest post hoc at a! = 0.05.
MUSCLE
R297
Pyruvate Oxidation
Kinetics. During recovery from burst exercise tissue [pyruvate] increases severalfold (32a). The effects of physiological changes in [pyruvate] on the oxidation of pyruvate, and consequently on lactate are shown in Fig. 1. At low respiration rates (state 4) the oxidative pathway
was saturated with pyruvate well below physiological levels, with an apparent Michaelis constant (Km) estimated to be 20fold and the kinetics showed marked changes. A much higher [pyruvate] was needed to saturate the oxidative pathway. The apparent Km increased to 37.4 t 2.5 (SE) PM. Comparisons with carp red muscle mitochondria were used to determine whether the pyruvate kinetics were a property of fish muscle or particular fiber type (Fig. 2). Carp red muscle in state 3 demonstrated kinetics similar to those observed in trout white muscle in state 4, i.e., l
min-’
.
120 100 state
4 (-ADP)
40 . 20 3
l
0
..I
.,
,
.
*.
a
@
f
120
i
100
F */+-----~ l
.-5 s.8 Jj 80
60
state 3 (+ADP)
40 20 80 Y
60
Stateb3+4OmMloctate
40
Fig. 1. Kinetics of pyruvate oxidation by trout white muscle mitochondria. Values are means k SE determined in lo-min incubations, expressed as percentage of rate determined with 1 mM pyruvate.Lowest concentration tested was 5 PM pyruvate. Absolute rates for each condition are as follows (means k SE in nmol [1-“C]pyruvate decarboxylated min-’ l mgprotein-‘):state 4 (top),O.82 * 0.08 (n = 5);state 3 (middle), 23.4 t 3.0 n = 4); state 3 with 40 mM lactate (bottom), 19.5 t 1.4 (n = 4). l
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R298
MITOCHONDRIAL
METABOLISM
@i? F 140 z = 120 8 c\l 100 75 80 s
.E 60 % g 40
carp
red State
muscle 3
3 20 . 5 St Q.
o0
0.1
0.2
0.3
[pyruvate]
0.4
0.5
(mtv!)
Fig. 2. Kinetics of carp red muscle mitochondrial pyruvate oxidation from 3 animals. Apparent K, was not determined but estimated to be below lowest [pyruvate] used (5 PM).
80 State
60
3
d
1
OF TROUT
WHITE
MUSCLE
However, at 20 PM pyruvate and high respiratory rates (state 3), fatty acid availability had no effect on pyruvate oxidation, which is consistent with pyruvate transport limiting pyruvate oxidation. The effects of fatty acid availability on PDHJCS are summarized in Table 1 for different respiratory states. Incubation in different states had little influence on PDHJCS (i.e., little covalent modification). Although there was no significant difference in PDHJCS in state 3 vs. 4 (Table l), there was a 95% reduction of PDH flux in intact mitochondria (Fig. 3). Thus most of the reduction of PDH flux observed in intact mitochondria in state 4 is due to allosteric inhibition of PDH, rather than covalent modification (changes between PDH, and PDHb). Similarly, incubation with 50 PM palmitoylcarnitine (in the presence of pyruvate) had no significant effect on PDH$CS, but PDH flux in isolated mitochondria decreased markedly (Fig. 3). Incubations with palmitoylcarnitine in the absence of pyruvate markedly inactivated PDH in situ (data not shown), confirming that the enzyme can also be covalently regulated. PDH Activity
During Recovery
J
PDH,/CS (Fig. 4) showed relatively little change with the exercise regime. A minor increase in activation state was observed immediately postexercise. During recovery, PDH, activity was relatively constant, near resting activities.
40 -
20 I/
0
I
incubation
time
5
10
15
20
(min)
Fig. 3. Effects of 50 PM palmitoylcamitine on pyruvate dehydrogenase flux. o and l , 20 PM pyruvate; A and A, 200 PM pyruvate; o and A, control incubations (-palmitoylcarnitine); l and A, incubations where 50 PM palmitoylcarnitine was added simultaneously. Rates obtained for 260 PM pyruvate controls are as follows (means k SE in nmol [ 1-“C]pyruvate decarboxylat&min-’ mg-‘: state 4 (left) 1.24 k 0.22 l
(n = 3);state3 (right),24.03k 2.58(n = 5).
not dependent on [pyruvate] over the physiological range. It has been reported that lactate competes for the rat heart mitochondrial pyruvate transporter, but with relatively low affinity (Km 12 mM, Ref. 12). Because fish develop extremely high postexercise lactate levels that remain for long periods, it was of interest to examine whether physiological lactate levels could inhibit pyruvate oxidation through transport competition. Pyruvate transport is potentially limiting only at high flux rates and low [pyruvate]. We examined the influence of 40 mM lactate under these conditions. No effects of 40 mM lactate on pyruvate oxidation were observed (Fig. 1). The apparent K, for pyruvate was 35.8 t 3.9 PM compared with 37.4 rf=2.5 PM in the absence of lactate. Effects of fatty acid oxidation. The effects of fatty acid availability were examined at high (200 PM) and low (20 PM) [pyruvate], each at high (state 3) and low (state 4) respiratory rates (Fig. 3). The control data also illustrate that the rate of pyruvate decarboxylation by isolated mitochondria was linear for the lO-min period used in the kinetics studies. PDH flux in isolated mitochondria was markedly reduced by fatty acid availability under most conditions.
Table 1. Effects of palmitoylcarnitine on PDH, in isolated trout white muscle mitochondria Palmitoylcamitine,
Aw PM
0
pM 60
300(state 3)
mU PDH,/mg 21.3k1.8 25.6k2.5 mU PDHJU CS 28.0 33.7 None (state 4) mU PDHJmg 15.5*1.7 19.4*3.5 mU PDHJU CS 20.4 25.5 Values are means zt SE for 5 animals; mU PDH,/U CS, (mean mU activated phosphate dehydrogenase/mg)/(0.76 U citrate synthaselmg). All incubations contained 200 /IM pyruvate. There was no significant effect of palmitoylcarnitine addition in state 3 or 4. There was no significant effect of respiratory state in the presence or absence of palmitoylcarnitine.
I
I
I
I,
1
R
Ex
2
4
8 recovery
I
L
n
I‘
81
I
24 time
(hr)
Fig. 4. Pyruvate dehydrogenase (PDH,) activation state in white muscle of burst-exercised rainbow trout. Activation state is expressed as mean ratio k SE of PDH, (mU)/citrate synthase (U) measured at 25OC. Number of animals at each time is reported with each data point. *Significantly different from rest.
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MITOCHONDRIAL
METABOLISM
PDH In Vitro vs. In Vivo The in vitro activity of PDH, in isolated mitochondria (Table 1) was close to that observed in resting fish in vivo (Fig. 4). Conversion between mitochondria rates and tissue activities must take into consideration both temperature differences and CS/mg mitochondrial protein. A mitochondrial PDH flux (15OC) of 10 nmol pyruvate/mg protein is equal to 13.16 nmol/U CS (0.76 U CS/mg at 15°C). The Q10 values for PDH, and CS are similar (1.48 and 1.52 respectively) so the effect of temperature on PDHJCS is negligible.
OF TROUT
WHITE
R299
MUSCLE
an ATP-producing capacity of 3.5 pmol ATP . mix?. assuming an ADP/O of 3.
g-l,
Effect of ATPIADP The results of our parallel study on changes in ATP/ ADPf (where ADPf is free ADP) in exercise and recovery are presented in Fig. 5. Several important features are obvious. At exhaustion there is a drop in ATP/ADPf by -50%. By 2 h, at which point phosphocreatine has recovered, ATP/ADPf has climbed to 2,000. By 8 h it is still elevated compared with rest but has recovered by 24 h.
Mitochondrial
Oxygen Consumption
Estimates of the maximal rates of substrate oxidation obtained with trout white muscle mitochondria are presented in Table 2. Pyruvate was oxidized at higher rates than fatty acids, but both substrates together elicited higher oxygen consumption rates than pyruvate alone. This may be related to the observation that palmitoylcarnitine oxidation in the presence of pyruvate did not covalently inactivate PDH. Tissue maximal oxygen consumption (Vo2 max) can be calculated from mitochondrial protein per gram of tissue (7.77 mg/g, Table 3) and the highest mitochondrial rate observed (75 nmolO2 min-’ mg-‘, Table 2) and is estimated at 583 nmol02.min-’ .g wet wt? This represents l
l
Table 2. Oxidation of physiological white muscle mitochondria
fuels by
trout
Substrate
nmol 02. min-’ . mg-’
Malate, 0.1 mM Pyruvate, 1 mM Palmitoylcarnitine, 50 PM Pyruvate + palmitoylcarnitine Lauroylcarnitine, 100 PM Octanoylcarnitine, 300 PM Acetylcarnitine, 1 mM RCR determinations (pyruvate + malate)
3.5kO.6
In the present study we examined the respiratory rates of mitochondria presented with physiological ATP/ADP (Fig. 5). We assume that the total ATP/ADP measured in isolated mitochondrial studies corresponds to ATP/ ADPf in vivo because the proteins, particularly myofibrils, that bind ADP in vivo are fractionated out of the preparation. At the high ratios that occur during recovery, it is apparent that very low rates of oxygen consumption would be elicited if this ratio were the sole controlling factor for mitochondrial respiration. At rest and exhaustion, ATP/ADPf is in the range where changes in the ratio would be expected to affect mitochondrial respiration (i.e., ~300). In the previous experiment Mg+ was omitted to allow establishment of high ATP/ADP. Mg+ addition so dramatically stimulated contaminating adenosinetriphosphatases (ATPases; i.e., possibly mitochondrial-bound hexokinase, myofibrillar or mitochondrial ATPases) that 2500
trout white muacle
66.5k4.9 39k3.5 75k6.3 37.5k3.0 35k4.1 37k2.6 77k5.6 3.5k0.25 22k1.9
state3 state4
11
recovery
cs
Tissue Mitochondria
25 25
PDH,
Mitochondria Mitochondria
25
15
Activity
n
U/g U/mg U/mg 27.9k2.7 mU/mg 18.9k1.7 mU/mg
34
9.32k0.67 1.2OkO.05 0.76kO.032
11
I1
1'
11
J
24 time
(h)
isolated mitochondria
a0 -
H Temp, ‘C
a
8
m i?! 603 z0 40-
Table 3. Activities of citrate synthuse and pyruvate dehydrogenase in trout white muscle and isolated mitochondria at 15 and 25OC Source
L
REx24
RCR Values are means =t SE for 5 animals determined at 15°C. All incubations contained 0.1 mM malate. State 4 rate is obtained after all the ADP is phosphorylated.
Enzyme
\
11 6 5 5
15 &lo ( 15-2S°C) 1.52t0.05 cs PDH, 1.48kO.06 PDH,/CS 0.97 Values are means * SE. CS, citrate synthase; PDH,, activated phosphate dehydrogenase. Mitochondrial protein/g tissue from (CS/ g)/(CS/mg mitochondrial protein) is 7.77 mg protein/g.
20-
O”“‘llll’llll’l’~‘J 0
100
200
300
400 500 600 700 800 AlP/ADP Fig. 5. Influence of ATP/ADP on trout white muscle mitochondrial respiration. Top: taken from mean values of ATP and calculated free ADP during recovery (32a). Estimates of tissue free ADP are calculated based on creatine kinase equilibrium, assuming all other reactants are free and homogeneously distributed within cell. Bottom: oxygen consumption of mitochondria isolated from trout white muscle exposed to variable ATP/ADP. With mitochondrial studies, it is assumed that all measured ADP is free, since proteins that bind ADP in vivo (myofibrils) are fractioned out during isolation. Each symbol types represents a different mitochondrial preparation.
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R300
MITOCHONDRIAL
METABOLISM
it was impossible to obtain high ATP/ADP in vitro. Because much of the ADPf present in the cell is bound to Mg+, we examined the effects of Mg+ on this mitochondrial preparation using a different protocol. If MgADP was the required substrate for the mitochondrial adenylate translocase, then a Mg+ dependence would be most pronounced under ADP-limiting conditions. Because there was no stimulation of ADP-limited respiration in the presence of 5 mM Mg+ (Table 4), we conclude that either the adenylate translocase does not discriminate between chelated and nonchelated ADP or adequate Mg+ was present as a contaminant. In any case the effects of ATP/ADP on mitochondrial respiration (Fig. 5) were apparently not influenced by our omission of physiological levels of Mg? The pattern of ATP/ADPf observed in exercised fish and the response of mitochondrial respiration to this ratio present a paradox. At a time when trout white muscle has an added metabolic cost (lactate metabolism), ATP/ADPf is more inhibitory to mitochondrial respiration than at rest. Further experiments indicated a potential role for phosphate and pH in stimulating respiration despite inhibitory ATP/ADPf.
OF TROUT
WHITE
MUSCLE
r 3.0 .-0 ‘;
L 0 \ E a
2.0
0 date 3 2.6
2.4
1””
0
fi
10
20
”
a
30
[phosphate]
40
(mM)
1
60 11111111.1 0
10
20
[phosphate]
30
40
(mM)
Fig. 6. Effects of phosphate concentration on mitochondrial respiration rate and ADP/O. State 3 incubations (0) contained 1 mM pyruvate and 0.1 mM malate and phosphate; 66% state 3 incubations (5 PM ADP, 0) were also given 100 U hexokinase, 5 mM MgCls, and 10 mM glucose. After a l- to 2-min preincubation, reactions were started with addition of ADP. In each case, 5 PM rates of oxygen consumption are corrected for ADP-independent respiration. Results are means & SE for 7 determinations. Left: ADP/O for state 3 incubations were calculated using known ADP addition and oxygen consumed between state 4 transitions. ADP/O for 5 PM ADP incubations were determined by measurement of glucose 6-phosphate produced in 5 min as described in MATERIALS AND METHODS. Right: respiration rates obtained as described above.
Effects of Phosphate Our phosphate experiments examined how physiological concentrations affect respiratory rate and efficiency of oxidative phosphorylation (as ADP/O). These data are ,presented in Fig. 6. The highest respiratory rates were observed over a range of 5-10 mM phosphate. At the highest concentration (40 mM), phosphate inhibited both the state 3 rate and the 66% state 3 rate (5 PM ADP) by 30%. Lower respiratory rates (70% of the 10 mM phosphate rate) were evident at 1 mM phosphate at both [ADP]. The efficiency of oxidative phosphorylation (ADP/O) was not affected by [phosphate] at either
NW*
The influence of pH on phosphate dependence is summarized in Fig. 7 and Table 5. The pH values were chosen to reflect rest (7.3) and early (6.5) and late (6.9) recovery (32a). At each [ADP] tested (2 and 0.5 PM ADP), acidosis decreased the sensitivity to phosphate. At each [ADP], the maximal respiration rates were achieved at lower [phosphate] in response to acidosis. There was a greater phosphate dependence at lower [ADP]. DISCUSSION
Carbon Metabolism
The first part of this study attempts to establish the importance of the dramatic changes in [lactate], [pyruTable
4. Mg*+ effects on respiratory properties
of the isolated trout white muscle mitochondria
Aw PM 300 (state 3) 5 2 Values are means it SE in tein-’ for 6 preparations. All and 0.1 mM malate.
Control
+5 mM MgC12
63k3.2 4Ok2.2 41k1.8 28A1.6 25k1.3 nmol 02*min-’ mg mitochondrial proincubations contained 1 mM pyruvate l
301 0
A 510 0 12 3 4 510 phosphate (mM) Fig. 7. Influence of pH on phosphate dependence of mitochondrial respiration. Mitochondria were incubated as described in Fig. 6 legend except that phosphate was excluded and ADP added at time0. Left: 2 PM ADP added; right: 0.5 PM ADP added. At l- to 2-min intervals small volumes (totaling
of trout white muscle:
D. MOYES, PATRICIA of Zoology, University of British
M. SCHULTE, Columbia,
Moyes, Christopher D., Patricia M. Schulte, and Peter W. Hochachka. Recovery metabolism of trout white muscle: role of mitochondria. Am. J. Physiol. 262 (Regulatory Integrative Comg. Physiol. 31): R295R304, 1992.-Recovery from burst exercise in fish is very slow. Lactate conversion to glycogenoccursprimarily within white muscleand must be fueled by mitochondrially produced ATP. In a parallel study we characterized the changes in tissue metabolites associatedwith burst exercise and recovery in rainbow trout (Oncorhynchus mykiss)white muscle.The present study examineswhether the mitochondrial capacity to produce ATP may limit the rate of recovery of trout white muscle.The cost (ATP l rnin-l *g-l) of glycogenresynthesis(0.05 pmol lactate converted* min-’ g tissue-l) was compared with the mitochondrial capacity to produce ATP. The cost of recovery can be met by only 3.5% of the maximal mitochondrial capacity. In fact, during recovery trout white musclemitochondria operate at a small fraction of their in vitro maximum. This capacity is suppressedin vivo by highly inhibitory ATP/ADP and limiting phosphate. The primary signal for increasedATP synthesisassociatedwith recovery is not a change in ATP/ADP but probably phosphate, elevated becauseof phosphocreatine hydrolysis and adenylate catabolism in the purine nucleotide cycle. At low ADP availability and suboptimal phosphate (6 mM), acidosisenhancesrespiration. At high respiratory rates mitochondrial pyruvate oxidation is sensitive to pyruvate concentration over the physiological range (apparent Michaelis constant = 35-40 PM). This sensitivity is lost at the low rates that approximate in vivo respiration. Changesin lactate do not affect the kinetics of pyruvate oxidation. Fatty acid oxidation may spare pyruvate and lactate for usein glyconeogenesis,primarily through allosteric inhibition of pyruvate dehydrogenaserather than covalent modification. l
AND
Vancouver,
PETER British
W. HOCHACHKA
Columbia
V6T 2A9, Canada
flounder recovery requires 8-12 h or more (19). Also, a number of indirect studies suggest that white muscle lactate is metabolized primarily in the white muscle. Injections of [“Cllactate into the blood remain primarily as lactate [skipjack tuna (39), flounder and salmon (19)]. Lactate turnover cannot account for the changes observed in white muscle lactate [salmon and flounder (19)], suggesting the blood and white muscle do not equilibrate. Studies using the glucose analogue deoxyglucose suggest that 40% of the glycogen resynthesized in white muscle after burst exercise in rainbow trout can be attributed to exogenous glucose uptake (T. G. West, P. M. Schulte, and P. W. Hochachka, unpublished observation). Furthermore, hepatocyte studies suggest that fish liver has insufficient capacity for gluconeogenesis from lactate to account for the rate of lactate disappearance (38). Because there is a net conservation of white muscle carbohydrate (glucosyl units + 2 X lactate) during exercise and recovery (32a), it is clear that oxidation is not an important route of lactate metabolism postexercise. The route for white muscle glycogen resynthesis from lactate is unclear. White muscle lacks adequate activities of pruvate carboxylase, one of the enzymes required for gluconeogenesis in liver (28). Alternate routes in mammals could involve malic enzyme plus phospho(enol)pyruvate carboxykinase (8) or reversal of pyruvate kinase (11). White muscle from most fish species lacks both pyruvate carboxylase and phospho(enoZ)pyruvate car-
boxykinase (e.g., Refs. 9,22), suggesting pyruvate kinase reversal may be the more important route of glyconeogenesis in fish. At present, direct evidence for any route ATP; ADP; phosphate; pyruvate; exercise; pH; purine nucleo- is lacking in fish. tide cycle Whatever the route of glycogen resynthesis, the ATP demands imposed by recovery metabolism (net glycogen HIGH-INTENSITY EXERCISE in mammalian white muscle is fueled by breakdown of glycogen, with formation of
lactate and accompanied by a tissue acidosis. Recovery metabolism involves reducing lactate concentration ([lactate]) to resting levels and repletion of glycogen stores. In mammals, if blood [lactate] is low, lactate is released and primarily oxidized in aerobic tissues, with a smaller proportion used as a glyconeogenic substrate (6). If perfusion [lactate] is high, conversion of lactate to glycogen within white muscle (glyconeogenesis) becomes increasingly important, especially in the more glycolytic fiber types (4, 29). Recovery from such exercise bouts typically takes several minutes to an hour (e.g., Ref. 15). Thus, in mammals, lactate metabolism postexercise is relatively fast and may involve several anabolic and catabolic tissues. Fish burst exercise and recovery differs from the mammalian pattern in a number of important aspects. The changes in metabolites are greater and more prolonged than in mammalian white muscle (19). Salmon and 0363-6119/92
$2.00 Copyright
synthesis) must be met by mitochondrial (aerobic) pathways. Comparison among the rates of recovery reported for various species [tuna > salmon > plaice (1, 21)]
reveals an apparent correlation with the mitochondrial density of the white muscle. Species with mitochondriarich white muscle recover faster than species with white muscle with low mitochondrial content. The present study investigates whether the ATP production by white muscle mitochondria limits the rate of glyconeogenesis. The cost of recovery is compared with mitochondrial capacity of the tissue. We also examine the influence of changes in ATP/ADP, pH, phosphate, and carbon metabolites on mitochondrial rate and substrate selection. MATERIALS AND METHODS Animals Rainbow trout (Oncorhynchus mykiss) of both sexes(400600 g) were held in continuously flowing fresh water at seasonal
temperatures. Animals were fed ad libitum four times a week.
0 1992 the American
Physiological
Society
R295
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R296
MITOCHONDRIAL
METABOLISM
OF TROUT
WHITE
MUSCLE
ExerciseProtocol
IsolatedMitochondrial Studies
The methodology involving exercise of fish is described in our parallel study (32a). Briefly, fish were exercisedto exhaustion in a swimtunnel and either killed immediately or allowed to recover for 2,4,8, or 24 h. An anesthetic (Somnotol) overdose technique was developed to kill the fish quickly with little struggling so asto minimize changesin labile metabolites.
Trout were killed by blow to the head. Approximately 40 g of white musclewascollected from an area extending from the dorsal fin to 3 cm anterior to the caudalpeduncle,including all tissue dorsal to the lateral line. After skinning the tissue,great care was taken to shear off any superficial red muscle.Tissue wasplaced on an ice-cold Petri dish, choppedwith razor blades, divided into three portions and addedto three Potter-Elvejhem homogenizers, each containing 30 ml isolation buffer. The composition of isolation buffer is as describedpreviously (23). Tissue was dispersedby two passesof a loosely fitting pestle followed by three passesof a more tightly fitting pestle. Homogenateswere combined and diluted to 500 ml with more isolation buffer and centrifuged 3 min at 2,660g. The supernatants were poured through four layers of cheeseclothand recentrifuged 5 min at 2,600 g. The supernatant was poured through eight layers of cheeseclothand centrifuged 10 min at 9,000g. Mitochondrial pellets were resuspendedin 10 ml isolation medium [minus bovine serum albumin (BSA)] and centrifuged 5 min at 8,000 g. The mitochondrial pellet was resuspended in l-2 ml BSA-free (to facilitate protein determinations) isolation medium and usedwithin 60 min.
Enzyme Activities Tissuehomogenateswere prepared in 9 vol of the following medium: 100 mM potassiumphosphate, 5 mM EDTA, 1 mM dichloroacetate, and 0.1% Triton X-100 at pH 7.0. Fluoride, often usedto inhibit pyruvate dehydrogenase-kinasewas not used as it inhibited pyruvate dehydrogenase(PDH) directly. Tissue was homogenizedby three bursts of an UltraTurrax grinder followed by three bursts of a Kontes sonicator. Homogenateswere centrifuged 10 min at 10,000g, with the resulting supernatantusedfor assaysof both phosphorylated active PDH (PDH,) and citrate synthase (CS). Activity of PDH, wasdetermined by measuringacetyl-CoAdependentacetylation of p-nitroaniline in the presenceof pigeonliver arylamine acetyltransferasepurified basedon Tabor et al. (36). Pigeon liver was frozen in liquid nitrogen and kept up to 2 mo before extraction. Tissue (100 g) was homogenized in 200 ml of 5 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-1 mM EDTA (pH 7.0). Homogenates were centrifuged 20 min at 12,006g. The pellets were rehomogenized in 200 ml of buffer and recentrifuged. The combined supernatantswere brought to 500 ml and addedto 400 ml icecold acetone. After centrifugation (10 min at 10,000g), the combinedsupernatantswere added to 1 liter acetone and centrifuged (5,000g for 10 min). The pellet wasdissolvedin 20 ml water for 60 min at 4°C and addedto 5 g alumina CT (pelleted from 50 ml suspension;Sigma Chemical). After 1 h of swirling (2 Hz), the suspensionwas centrifuged 7 min at 1,000g. The pellet waswashedtwice with 100 ml water before elution with 250 ml potassiumphosphatebuffer (100 mM, pH 7.7) in four aliquots. The enzyme wasconcentrated by Amicon Centriprep units and frozen (080°C) until needed.Each purification was checked for contaminating PDH, activity, and none was detected. PDH, activity in tissueextracts was assayedusing a PerkinElmer Lambda 2 spectrophotometer, interfaced with an IBM computer using PECSS (Perkin-Elmer) software to calculate enzyme activities. The l-ml assay mixture contained 1 mM MgCls, 0.5 mM EDTA, 0.5 mM cocarboxylase, 0.15 mM CoA, 2 mM NAD+, 5 mM /3-mercaptoethanol,0.1 mMp-nitroaniline, 200 mU a&mine acetyltransferase, 40 ~1 homogenate, and 100mM tris(hydroxymethyl)aminomethane (Tris; pH 7.7). Reaction was initiated by addition of 1 mM pyruvate and was linear for at least5 min. No activity wasdetectedin the absence of pyruvate. Activity was measuredwithin 30 min of homogenization. Attempts to fully activate fish musclePDH by purified pig heart PDH-phosphatasewere unsuccessful.To standardize for interindividual differences in mitochondrial density, PDH, activity is expressedagainst CS activity determined in the same homogenate. The CS assaycontained 0.3 mM acetyl-CoA, 0.2 mM 5,5’dithio-bis(2nitrobenzoic acid), and 100 mM Tris (pH 8.0) and was initiated by addition of 0.5 mM oxaloacetate. Blank rates (minus oxaloacetate)were ~5% of the CS rate. All enzyme assayson whole tissue extracts were performed at 25°C to obtain maximal sensitivity of the PDH, assay and minimize coupling enzyme requirements. Q10determinations were performed to allow conversion between tissue enzyme analysesand mitochondrial studiesperformed at l5OC.
Oxygen Consumption Determination of the rate of oxidation of physiological substrates was as describedpreviously (24) at pH 7.3 and 15OC. The state 3 rate is the rate of oxygen consumption in the presenceof ADP (0.3 mM). The state 4 rate is obtained after all the ADP hasbeen phosphorylated. The respiratory control ratio (RCR) equalsstate 3 divided by state 4. The oxygen consumption rate was also determined as a function of the ratio ATP/ADP. Assays were performed with 0.3-0.5 mg protein/ml assay medium. Injections of 7.5 mM ATP, 10 mM glucose,then increasing hexokinase levels were used to obtain an ATP/ADP closeto that seenin recovering fish (32a). Aliquots (500 ~1) were removed from parallel incubations and added to 500 ~1ethanol for deproteinization. The supernatants (10 min at 15,000 g) were frozen for ATP and ADP determinations by high-performance liquid chromatography (32a). The influence of MgC12was determined at subsaturating [ADP]. Mitochondria were addedto a cuvette containing assay medium (minus BSA), 1 mM pyruvate, 0.1 mM malate, and 150 U hexokinase with and without 5 mM MgClz. The high activity of hexokinase was usedto preclude an indirect effect of M$+ due to Mg2+-dependent stimulation of hexokinase. Reactions were started by addition of 5 or 2 PM ADP. The ADP levels were chosen to elicit submaximal rates that were dependent on ADP transport rate into the mitochondria, analogousto high ATP/ADP. The actual ATP/ADP wasnot determined. In experiments where we used a hexokinase ADP-regenerating system, ATP production rates were determined by assaying glucose6-phosphateplus fructose 6-phosphateproduction. Because such high activities of hexokinase were used, the relatively low contamination by phosphoglucoisomerase became significant. Approximately 10% of the glucose6=phosphate was converted to fructose 6-phosphate. Mitochondrial incubations (0.9 ml) were addedto 0.1 ml 70% perchloric acid and centrifuged 5 min at 15,060g. Aliquots of the supematant were neutralized by addition of 1 M Tris (40 mM final concn) and 10 M KOH. Glucose6-phosphateplus fructose g-phosphate was assayedin 50 mM Tris (pH 8.0) and 1 mM NADP+. The reaction was started by addition of 1 U glucose-6-phosphate dehydrogenaseplus 1 U phosphoglucoisomerase and was complete within 2 min.
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MITOCHONDRIAL
METABOLISM
OF TROUT
WHITE
Mitochondrial Pyruvate Oxidation
RESULTS
Pyruvate oxidation was assayedusing [1-14C]pyruvate. Becausethe radiolabeledcarbon is removed in the decarboxylation reaction of the PDH complex, “CO2 production representsthe flux through PDH in situ (within mitochondria). This technique was used to examine the influence of a number of potential effecters of mitochondrial metabolism. Also, some comparisonswere performed between mitochondria from trout white muscle and carp red muscle. Carp mitochondria were preparedaccordingto Moyes et al. (23) and assayedasdescribed here for the trout preparation. Small volumes of mitochondria were added to the assay medium used for oxygen consumption experiments to a total volume of 2 ml. Incubations were performed in 20-ml scintillation vials. A 5-min preincubation period was sufficient time to allow the vials to be cappedwith rubber stoppersfitted with plastic center wells. Reaction was started by injection of 100 ~1 of pyruvate (12C+ 14C,0.25 &i//rmol final sp act at each pyruvate concn) through the rubber cap, and vials were shaken (1 Hz) at 15OCfor 10 min unlessnoted otherwise. Incubations were terminated by a 200.~1injection of 70% perchloric acid. Hyamine hydroxide (150 ~1) was addedto the center well, and vials were shaken90 min at room temperature to collect 14C02. Filter paperswere addedto 10 ml of ACS-2 (Amersham), with 0.1% acetic acid to prevent background color formation and eliminate chemiluminescence.Sampleswere counted for 10min in a LKB 1214 RackBeta scintillation counter with programmedquench correction. All assayswere done in at least duplicate. A similar protocol was used to determine PDH, activities with solubilized mitochondria. The assay componentswere as describedfor tissue PDH, anaIysis except p-nitroaniline and a&mine acetyltransferasewere omitted and coenzyme A was elevated to 0.6 mM to prevent significant depletion in the longer incubations. Decarboxylation of [l-14C]pyruvate was measuredasbefore. The reaction was shown to be linear for at least 10 min using PDH that was inactivated by 20-min incubation of mitochondria with 50 PM palmitoylcarnitine. The linearity of the PDH, assay with inactivated PDH demonstrates that the assay procedure did not allow activation in vitro. Routinely, assay incubations were run for 6 min in at least duplicate. Studies examining the kinetics of pyruvate oxidation by intact mitochondria were performed under the conditions described above. Oxygen consumption rates provided estimates of pyruvate oxidation, which were usedto establishthe highest mitochondrial concentration that would not deplete pyruvate by X0% at the lowest [pyruvate] usedin the radiolabel studies. Preliminary studiesshowedthe rate of pyruvate oxidation was linear with respect to [protein] over the ranges used in this study and linear for at least 15 min. The influence of ADP availability was examined under two conditions. Experiments in state 4 (no added ADP) used high [protein] to obtain adequatedisintegrations-per-minute collection. State 3 experiments (+ADP) wereperformed with 0.3 mM ADP, which was not substantially depleted becausemitochondria were diluted and incubations were limited to 10 min. In someexperiments lactate (40 mM) was added5 min before addition of pyruvate.
Mitochondrial
Statistics Where statistical comparisonswere required, an analysis of variance was usedwith Tukey’s multiple comparisontest post hoc at a! = 0.05.
MUSCLE
R297
Pyruvate Oxidation
Kinetics. During recovery from burst exercise tissue [pyruvate] increases severalfold (32a). The effects of physiological changes in [pyruvate] on the oxidation of pyruvate, and consequently on lactate are shown in Fig. 1. At low respiration rates (state 4) the oxidative pathway
was saturated with pyruvate well below physiological levels, with an apparent Michaelis constant (Km) estimated to be 20fold and the kinetics showed marked changes. A much higher [pyruvate] was needed to saturate the oxidative pathway. The apparent Km increased to 37.4 t 2.5 (SE) PM. Comparisons with carp red muscle mitochondria were used to determine whether the pyruvate kinetics were a property of fish muscle or particular fiber type (Fig. 2). Carp red muscle in state 3 demonstrated kinetics similar to those observed in trout white muscle in state 4, i.e., l
min-’
.
120 100 state
4 (-ADP)
40 . 20 3
l
0
..I
.,
,
.
*.
a
@
f
120
i
100
F */+-----~ l
.-5 s.8 Jj 80
60
state 3 (+ADP)
40 20 80 Y
60
Stateb3+4OmMloctate
40
Fig. 1. Kinetics of pyruvate oxidation by trout white muscle mitochondria. Values are means k SE determined in lo-min incubations, expressed as percentage of rate determined with 1 mM pyruvate.Lowest concentration tested was 5 PM pyruvate. Absolute rates for each condition are as follows (means k SE in nmol [1-“C]pyruvate decarboxylated min-’ l mgprotein-‘):state 4 (top),O.82 * 0.08 (n = 5);state 3 (middle), 23.4 t 3.0 n = 4); state 3 with 40 mM lactate (bottom), 19.5 t 1.4 (n = 4). l
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R298
MITOCHONDRIAL
METABOLISM
@i? F 140 z = 120 8 c\l 100 75 80 s
.E 60 % g 40
carp
red State
muscle 3
3 20 . 5 St Q.
o0
0.1
0.2
0.3
[pyruvate]
0.4
0.5
(mtv!)
Fig. 2. Kinetics of carp red muscle mitochondrial pyruvate oxidation from 3 animals. Apparent K, was not determined but estimated to be below lowest [pyruvate] used (5 PM).
80 State
60
3
d
1
OF TROUT
WHITE
MUSCLE
However, at 20 PM pyruvate and high respiratory rates (state 3), fatty acid availability had no effect on pyruvate oxidation, which is consistent with pyruvate transport limiting pyruvate oxidation. The effects of fatty acid availability on PDHJCS are summarized in Table 1 for different respiratory states. Incubation in different states had little influence on PDHJCS (i.e., little covalent modification). Although there was no significant difference in PDHJCS in state 3 vs. 4 (Table l), there was a 95% reduction of PDH flux in intact mitochondria (Fig. 3). Thus most of the reduction of PDH flux observed in intact mitochondria in state 4 is due to allosteric inhibition of PDH, rather than covalent modification (changes between PDH, and PDHb). Similarly, incubation with 50 PM palmitoylcarnitine (in the presence of pyruvate) had no significant effect on PDH$CS, but PDH flux in isolated mitochondria decreased markedly (Fig. 3). Incubations with palmitoylcarnitine in the absence of pyruvate markedly inactivated PDH in situ (data not shown), confirming that the enzyme can also be covalently regulated. PDH Activity
During Recovery
J
PDH,/CS (Fig. 4) showed relatively little change with the exercise regime. A minor increase in activation state was observed immediately postexercise. During recovery, PDH, activity was relatively constant, near resting activities.
40 -
20 I/
0
I
incubation
time
5
10
15
20
(min)
Fig. 3. Effects of 50 PM palmitoylcamitine on pyruvate dehydrogenase flux. o and l , 20 PM pyruvate; A and A, 200 PM pyruvate; o and A, control incubations (-palmitoylcarnitine); l and A, incubations where 50 PM palmitoylcarnitine was added simultaneously. Rates obtained for 260 PM pyruvate controls are as follows (means k SE in nmol [ 1-“C]pyruvate decarboxylat&min-’ mg-‘: state 4 (left) 1.24 k 0.22 l
(n = 3);state3 (right),24.03k 2.58(n = 5).
not dependent on [pyruvate] over the physiological range. It has been reported that lactate competes for the rat heart mitochondrial pyruvate transporter, but with relatively low affinity (Km 12 mM, Ref. 12). Because fish develop extremely high postexercise lactate levels that remain for long periods, it was of interest to examine whether physiological lactate levels could inhibit pyruvate oxidation through transport competition. Pyruvate transport is potentially limiting only at high flux rates and low [pyruvate]. We examined the influence of 40 mM lactate under these conditions. No effects of 40 mM lactate on pyruvate oxidation were observed (Fig. 1). The apparent K, for pyruvate was 35.8 t 3.9 PM compared with 37.4 rf=2.5 PM in the absence of lactate. Effects of fatty acid oxidation. The effects of fatty acid availability were examined at high (200 PM) and low (20 PM) [pyruvate], each at high (state 3) and low (state 4) respiratory rates (Fig. 3). The control data also illustrate that the rate of pyruvate decarboxylation by isolated mitochondria was linear for the lO-min period used in the kinetics studies. PDH flux in isolated mitochondria was markedly reduced by fatty acid availability under most conditions.
Table 1. Effects of palmitoylcarnitine on PDH, in isolated trout white muscle mitochondria Palmitoylcamitine,
Aw PM
0
pM 60
300(state 3)
mU PDH,/mg 21.3k1.8 25.6k2.5 mU PDHJU CS 28.0 33.7 None (state 4) mU PDHJmg 15.5*1.7 19.4*3.5 mU PDHJU CS 20.4 25.5 Values are means zt SE for 5 animals; mU PDH,/U CS, (mean mU activated phosphate dehydrogenase/mg)/(0.76 U citrate synthaselmg). All incubations contained 200 /IM pyruvate. There was no significant effect of palmitoylcarnitine addition in state 3 or 4. There was no significant effect of respiratory state in the presence or absence of palmitoylcarnitine.
I
I
I
I,
1
R
Ex
2
4
8 recovery
I
L
n
I‘
81
I
24 time
(hr)
Fig. 4. Pyruvate dehydrogenase (PDH,) activation state in white muscle of burst-exercised rainbow trout. Activation state is expressed as mean ratio k SE of PDH, (mU)/citrate synthase (U) measured at 25OC. Number of animals at each time is reported with each data point. *Significantly different from rest.
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MITOCHONDRIAL
METABOLISM
PDH In Vitro vs. In Vivo The in vitro activity of PDH, in isolated mitochondria (Table 1) was close to that observed in resting fish in vivo (Fig. 4). Conversion between mitochondria rates and tissue activities must take into consideration both temperature differences and CS/mg mitochondrial protein. A mitochondrial PDH flux (15OC) of 10 nmol pyruvate/mg protein is equal to 13.16 nmol/U CS (0.76 U CS/mg at 15°C). The Q10 values for PDH, and CS are similar (1.48 and 1.52 respectively) so the effect of temperature on PDHJCS is negligible.
OF TROUT
WHITE
R299
MUSCLE
an ATP-producing capacity of 3.5 pmol ATP . mix?. assuming an ADP/O of 3.
g-l,
Effect of ATPIADP The results of our parallel study on changes in ATP/ ADPf (where ADPf is free ADP) in exercise and recovery are presented in Fig. 5. Several important features are obvious. At exhaustion there is a drop in ATP/ADPf by -50%. By 2 h, at which point phosphocreatine has recovered, ATP/ADPf has climbed to 2,000. By 8 h it is still elevated compared with rest but has recovered by 24 h.
Mitochondrial
Oxygen Consumption
Estimates of the maximal rates of substrate oxidation obtained with trout white muscle mitochondria are presented in Table 2. Pyruvate was oxidized at higher rates than fatty acids, but both substrates together elicited higher oxygen consumption rates than pyruvate alone. This may be related to the observation that palmitoylcarnitine oxidation in the presence of pyruvate did not covalently inactivate PDH. Tissue maximal oxygen consumption (Vo2 max) can be calculated from mitochondrial protein per gram of tissue (7.77 mg/g, Table 3) and the highest mitochondrial rate observed (75 nmolO2 min-’ mg-‘, Table 2) and is estimated at 583 nmol02.min-’ .g wet wt? This represents l
l
Table 2. Oxidation of physiological white muscle mitochondria
fuels by
trout
Substrate
nmol 02. min-’ . mg-’
Malate, 0.1 mM Pyruvate, 1 mM Palmitoylcarnitine, 50 PM Pyruvate + palmitoylcarnitine Lauroylcarnitine, 100 PM Octanoylcarnitine, 300 PM Acetylcarnitine, 1 mM RCR determinations (pyruvate + malate)
3.5kO.6
In the present study we examined the respiratory rates of mitochondria presented with physiological ATP/ADP (Fig. 5). We assume that the total ATP/ADP measured in isolated mitochondrial studies corresponds to ATP/ ADPf in vivo because the proteins, particularly myofibrils, that bind ADP in vivo are fractionated out of the preparation. At the high ratios that occur during recovery, it is apparent that very low rates of oxygen consumption would be elicited if this ratio were the sole controlling factor for mitochondrial respiration. At rest and exhaustion, ATP/ADPf is in the range where changes in the ratio would be expected to affect mitochondrial respiration (i.e., ~300). In the previous experiment Mg+ was omitted to allow establishment of high ATP/ADP. Mg+ addition so dramatically stimulated contaminating adenosinetriphosphatases (ATPases; i.e., possibly mitochondrial-bound hexokinase, myofibrillar or mitochondrial ATPases) that 2500
trout white muacle
66.5k4.9 39k3.5 75k6.3 37.5k3.0 35k4.1 37k2.6 77k5.6 3.5k0.25 22k1.9
state3 state4
11
recovery
cs
Tissue Mitochondria
25 25
PDH,
Mitochondria Mitochondria
25
15
Activity
n
U/g U/mg U/mg 27.9k2.7 mU/mg 18.9k1.7 mU/mg
34
9.32k0.67 1.2OkO.05 0.76kO.032
11
I1
1'
11
J
24 time
(h)
isolated mitochondria
a0 -
H Temp, ‘C
a
8
m i?! 603 z0 40-
Table 3. Activities of citrate synthuse and pyruvate dehydrogenase in trout white muscle and isolated mitochondria at 15 and 25OC Source
L
REx24
RCR Values are means =t SE for 5 animals determined at 15°C. All incubations contained 0.1 mM malate. State 4 rate is obtained after all the ADP is phosphorylated.
Enzyme
\
11 6 5 5
15 &lo ( 15-2S°C) 1.52t0.05 cs PDH, 1.48kO.06 PDH,/CS 0.97 Values are means * SE. CS, citrate synthase; PDH,, activated phosphate dehydrogenase. Mitochondrial protein/g tissue from (CS/ g)/(CS/mg mitochondrial protein) is 7.77 mg protein/g.
20-
O”“‘llll’llll’l’~‘J 0
100
200
300
400 500 600 700 800 AlP/ADP Fig. 5. Influence of ATP/ADP on trout white muscle mitochondrial respiration. Top: taken from mean values of ATP and calculated free ADP during recovery (32a). Estimates of tissue free ADP are calculated based on creatine kinase equilibrium, assuming all other reactants are free and homogeneously distributed within cell. Bottom: oxygen consumption of mitochondria isolated from trout white muscle exposed to variable ATP/ADP. With mitochondrial studies, it is assumed that all measured ADP is free, since proteins that bind ADP in vivo (myofibrils) are fractioned out during isolation. Each symbol types represents a different mitochondrial preparation.
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R300
MITOCHONDRIAL
METABOLISM
it was impossible to obtain high ATP/ADP in vitro. Because much of the ADPf present in the cell is bound to Mg+, we examined the effects of Mg+ on this mitochondrial preparation using a different protocol. If MgADP was the required substrate for the mitochondrial adenylate translocase, then a Mg+ dependence would be most pronounced under ADP-limiting conditions. Because there was no stimulation of ADP-limited respiration in the presence of 5 mM Mg+ (Table 4), we conclude that either the adenylate translocase does not discriminate between chelated and nonchelated ADP or adequate Mg+ was present as a contaminant. In any case the effects of ATP/ADP on mitochondrial respiration (Fig. 5) were apparently not influenced by our omission of physiological levels of Mg? The pattern of ATP/ADPf observed in exercised fish and the response of mitochondrial respiration to this ratio present a paradox. At a time when trout white muscle has an added metabolic cost (lactate metabolism), ATP/ADPf is more inhibitory to mitochondrial respiration than at rest. Further experiments indicated a potential role for phosphate and pH in stimulating respiration despite inhibitory ATP/ADPf.
OF TROUT
WHITE
MUSCLE
r 3.0 .-0 ‘;
L 0 \ E a
2.0
0 date 3 2.6
2.4
1””
0
fi
10
20
”
a
30
[phosphate]
40
(mM)
1
60 11111111.1 0
10
20
[phosphate]
30
40
(mM)
Fig. 6. Effects of phosphate concentration on mitochondrial respiration rate and ADP/O. State 3 incubations (0) contained 1 mM pyruvate and 0.1 mM malate and phosphate; 66% state 3 incubations (5 PM ADP, 0) were also given 100 U hexokinase, 5 mM MgCls, and 10 mM glucose. After a l- to 2-min preincubation, reactions were started with addition of ADP. In each case, 5 PM rates of oxygen consumption are corrected for ADP-independent respiration. Results are means & SE for 7 determinations. Left: ADP/O for state 3 incubations were calculated using known ADP addition and oxygen consumed between state 4 transitions. ADP/O for 5 PM ADP incubations were determined by measurement of glucose 6-phosphate produced in 5 min as described in MATERIALS AND METHODS. Right: respiration rates obtained as described above.
Effects of Phosphate Our phosphate experiments examined how physiological concentrations affect respiratory rate and efficiency of oxidative phosphorylation (as ADP/O). These data are ,presented in Fig. 6. The highest respiratory rates were observed over a range of 5-10 mM phosphate. At the highest concentration (40 mM), phosphate inhibited both the state 3 rate and the 66% state 3 rate (5 PM ADP) by 30%. Lower respiratory rates (70% of the 10 mM phosphate rate) were evident at 1 mM phosphate at both [ADP]. The efficiency of oxidative phosphorylation (ADP/O) was not affected by [phosphate] at either
NW*
The influence of pH on phosphate dependence is summarized in Fig. 7 and Table 5. The pH values were chosen to reflect rest (7.3) and early (6.5) and late (6.9) recovery (32a). At each [ADP] tested (2 and 0.5 PM ADP), acidosis decreased the sensitivity to phosphate. At each [ADP], the maximal respiration rates were achieved at lower [phosphate] in response to acidosis. There was a greater phosphate dependence at lower [ADP]. DISCUSSION
Carbon Metabolism
The first part of this study attempts to establish the importance of the dramatic changes in [lactate], [pyruTable
4. Mg*+ effects on respiratory properties
of the isolated trout white muscle mitochondria
Aw PM 300 (state 3) 5 2 Values are means it SE in tein-’ for 6 preparations. All and 0.1 mM malate.
Control
+5 mM MgC12
63k3.2 4Ok2.2 41k1.8 28A1.6 25k1.3 nmol 02*min-’ mg mitochondrial proincubations contained 1 mM pyruvate l
301 0
A 510 0 12 3 4 510 phosphate (mM) Fig. 7. Influence of pH on phosphate dependence of mitochondrial respiration. Mitochondria were incubated as described in Fig. 6 legend except that phosphate was excluded and ADP added at time0. Left: 2 PM ADP added; right: 0.5 PM ADP added. At l- to 2-min intervals small volumes (totaling
