1 Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom; and 2 Division of Biochemistry, University of Tasmania, Hobart 7001, Tasmania, Australia
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ABSTRACT |
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Proton pumping across the mitochondrial inner membrane and proton leak back through the natural proton conductance pathway make up a futile cycle that dissipates redox energy. We measured respiration and average mitochondrial membrane potential in perfused rat hindquarter with maximal tetanic contraction of the left gastrocnemius-soleus-plantaris muscle group, and we estimate that the mitochondrial proton cycle accounted for 34% of the respiration rate of the preparation. Similar measurements in rat hepatocytes given substrates to cause a high rate of gluconeogenesis and ureagenesis showed that the proton cycle accounted for 22% of the respiration rate of these cells. Combining these in vitro values with literature values for the contribution of skeletal muscle and liver to standard metabolic rate (SMR), we calculate that the proton cycle in working muscle and liver may account for 15% of SMR in vivo. Although this value is less than the 20% of SMR we calculated previously using data from resting skeletal muscle and hepatocytes, it is still large, and we conclude that the futile proton cycle is a major contributor to SMR.
standard metabolic rate; proton pumping; redox energy; contracting skeletal muscle; working liver cells
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INTRODUCTION |
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STANDARD METABOLIC RATE (SMR; also termed basal metabolic rate) is the steady-state rate of heat production by a whole organism under a set of standard conditions. For mammals, these conditions are that the individual is awake but resting, stress free, not digesting food (prior food intake being at or around maintenance level) and maintained at a thermoneutral temperature. SMR is either measured directly as heat production or indirectly as oxygen consumption, from which it can be accurately predicted.
Mammals oxidize substrates at a considerable rate in the standard state, when no net work is done and all the free energy is dissipated. The question of which futile cycles are driven by respiration has been the subject of much research effort. Recent attempts to quantify the contribution of the cellular processes that underlie SMR indicate that the cycles catalyzed by the Na+-K+-ATPase and plasma membrane monovalent cation conductance pathways, Ca2+-ATPase and Ca2+ conductance pathways, and protein synthesis and degradation together account for ~40% of SMR in adult rats (see Refs. 8, 25, 29 for review). In addition, we recently showed (4, 24) that a futile cycle of proton extrusion across the mitochondrial inner membrane and the subsequent proton leak back to the matrix via endogenous proton conductance pathways accounted for about one-half of the oxygen consumption rate of resting, perfused rat skeletal muscle (24) and one-fourth of the oxygen consumption rate of resting isolated liver cells (4). Using these data, we calculated that, in vivo, the proton leak pathway in liver and skeletal muscle alone could account for one-fifth of rat SMR. When other tissues are included in this calculation, the proton cycle could account for 25% of SMR (24).
However, there are difficulties in extrapolating the data in Rolfe and Brand (24) to whole-animal SMR. Resting perfused skeletal muscle and isolated liver cells may have a lower ATP demand than under standard conditions in vivo. For example, skeletal muscle in a resting but awake rat will contract to maintain muscle tone and posture, and the liver may have some ATP demand for pathways such as the synthesis of urea and glucose, but these processes may have been partially or completely absent in our resting preparations. An unphysiologically low ATP demand might cause the contribution of proton leak to metabolic rate to be overestimated. There are two reasons for this. First, if ATP demand is low, then the contribution of proton leak will be proportionately increased. Second, if ATP demand is low, its driving force, the mitochondrial protonmotive force, will increase and drive the proton leak faster.
In the present paper, we set the ATP demand of our model systems to give double the resting respiration rates by inducing contraction in perfused skeletal muscle and by incubating the liver cells in a medium that stimulates gluconeogenesis and ureagenesis. We have measured the rate of the proton cycle under these new conditions and estimated its contribution to the oxygen consumption rate of skeletal muscle and liver under standard conditions in vivo.
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EXPERIMENTAL PROCEDURES |
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Isolation and perfusion of the rat hindquarter.
Male Hooded Wistar rats (~2 mo old, 180-220 g body wt) were
maintained at a thermoneutral temperature (25°C) and had access ad
libitum to a complete diet (Gibsons, Hobart, Australia) and drinking
water. They were anesthetized with sodium pentobarbitone (60 mg/kg body
mass ip), and the hindquarter was isolated from the rest of the
vascular system of the rat essentially according to the method of
Ruderman et al. (26), with additional modifications as described by
Colquhoun et al. (9), the procedure taking ~20 min up to the
cannulation of the vena cava and aorta. The resting hindquarter was
perfused at a constant flow rate (0.5 ml · g
muscle1 · min
1)
with buffer containing (in mM) 118 NaCl, 5 KCl, 1 KH2PO4,
1.18 MgSO4, 25 NaHCO3, 8.3 glucose, 2.5 CaCl2, and 1.2 sodium pyruvate, together with 4.6% (wt/vol) BSA, 0.46 U/ml sodium heparin, and 35%
(vol/vol) bovine red blood cells [isolated essentially as described by Dora et al. (10)]. All perfusions were carried out
in a temperature-controlled cabinet maintained at 32°C. The perfusate was gassed with 95% air-5%
CO2 (vol/vol) via a Silastic tube
oxygenator, and the temperature was brought to 32°C by passage through a heat exchanger coil. About 200 ml of perfusate were allowed
to flow to waste, and then the effluent was connected to a reservoir of
300 ml of perfusate containing 1 nM (45 nCi/ml) [3H]methyltriphenylphosphonium
cation ([3H]TPMP). The
hindquarter was then perfused using this closed, recirculating system
for the remainder of the experiment. Perfusion pressure was monitored
via a side-arm proximal to the aorta.
Induction of contraction in the isolated hindquarter.
The skin was removed from the thigh of the left limb, and the sciatic
nerve was exposed in the flank and cut to allow the positioning
of the distal cut end in a suction electrode. The knee was secured
by the tibiopatellar ligament, and the Achilles tendon was attached to
a Harvard Apparatus isometric transducer, thereby allowing transmission
of tension from the gastrocnemius-plantaris-soleus muscle group. The
perfusion rate was then increased to 1 ml · min1 · g
muscle
1, and, 10 min after
this flow rate increase, stimulation of the sciatic nerve was initiated
and the resting length of the muscle was adjusted to attain maximal
active tension on stimulation (to get maximum active tension, twitch
stimulations of 10 ms in duration were used). The oxygen consumption
rate of the hindquarter was monitored as described below to assess the
effect of the flow rate changes on the respiration rate of the
preparation. We found no significant difference in the rate of
hindquarter oxygen consumption before and after the flow rate changes
(0.358 ± 0.07 and 0.362 ± 0.04 µmol
O2 · min
1 · g
tissue
1, respectively;
means ± SE; n = 5). Tension
development was recorded during contraction by a recording oscilloscope
(Telequipment model DM64) and by a Yokagawa 3056 chart recorder.
Tetanic stimulation involved 200-ms trains of 100 Hz with 0.1-ms pulses
applied every 2 s, adjusted (3-5 V) to attain full fiber
recruitment (22). Figure 1
shows typical graphs of tension and oxygen consumption and of tension
and membrane potential.
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Measurement of hindquarter oxygen consumption and electrochemical
potentials.
Arterial and venous perfusate samples were taken using gastight 1-ml
syringes and stored on ice until they were analyzed for total oxygen
content using a galvanic cell oxygen analyzer (TasCon oxygen content
analyzer, manufactured by the Physiology Department, University of
Tasmania). Two arterial samples were taken (one at the 0.5 ml · min1 · g
tissue
1 flow rate and one
at 1 ml · min
1 · g
tissue
1) and up to eight
venous samples (two at 0.5 ml · min
1 · g
tissue
1, one or two
5-10 min after increase of flow to 1 ml · min
1 · g
tissue
1 before contraction
was induced, two after contraction was initiated, two after oligomycin
was added, and two after cyanide addition). Duplicate analysis of each
collected sample took an average of 15 min (20). Measurement of tissue
membrane potential was achieved using
[3H]TPMP as described
previously (24). Samples (1 ml) were taken from the reservoir before
and at various stages during the perfusion. Each sample was spun at
16,000 g in an Anderman bench
centrifuge to pellet the erythrocytes, and duplicate supernatant
samples (200 µl) were taken. Each sample was dispersed in 3.5 ml of
scintillant (BCS, Amersham International, Amersham, Buckinghamshire,
UK), and radioactivity was determined by counting in a liquid
scintillation counter (LS 3801, Beckman Instruments, Irvine, CA) with
corrections for quench. The amount of radioisotope taken up by the
tissue was calculated as the total activity of the isotope added to the perfusate (measured by sampling the perfusate at the time the recirculating perfusion was started) less the total activity remaining in the perfusate once a steady-state value for the TPMP was established (~100 min). The average mitochondrial membrane potential (
) was
calculated as described previously (24), except that the plasma
membrane potential was assumed to drop by 20 mV (15) in 15% of the
preparation during contraction.
Experiments using liver cells.
Female Wistar rats (~220-260 g) were starved overnight.
Hepatocytes were prepared in the absence of glucose as described in Refs. 18 and 27 and kept in a conical flask on ice for up to 3 h before
use. Measurements of viability (94.9 ± 1.2%), dry cell mass,
respiration, and in situ (using
[3H]TPMP) and
incubation protocols were as described in Refs. 3 and 12. Cells were
taken from the stock solution on ice and diluted fivefold to 27.9 ± 1.0 mg dry mass/ml into 2 ml of medium in 20-ml glass vials maintained
at 37°C in a shaking water bath and then gassed with 95% air-5%
CO2. The medium contained (in mM)
106 NaCl, 5 KCl, 25 NaHCO3, 0.41 MgSO4, 10 NaHPO4, 2.5 CaCl2, 10 sodium
D-lactate, 1 sodium pyruvate, 10 L-glutamine, and 2 L-ornithine, together with
2.25% (wt/vol) defatted BSA, 1 µM TPMP, and 100 µg/ml inulin (pH
7.2). After 10 min of preincubation, appropriate radioisotopes and
inhibitors were added (oligomycin, myxothiazol, 0.8 µCi
3H2O,
and 0.04 µCi
[14C]methoxyinulin or
0.08 µCi
3H[TPMP]);
20 min later, duplicate incubations were transferred to oxygen
electrodes for measurements of respiration rate and triplicate samples
from one incubation were transferred to microcentrifuge tubes and
centrifuged, for radioactivity measurements for cell volume (1.39 ± 0.06 µl/mg dry mass) and
(3). Potentials were calculated from
measured TPMP uptake and cell volume as in Ref. 3, under the assumption
that 19% of the volume of rat hepatocytes is occupied by mitochondria
(12, 17, 18), with TPMP binding corrections of 0.44 in the
mitochondrial matrix (19, 20), 0.20 in the cytoplasm (12, 17), and 0.71 in the medium (17) and a value for plasma membrane potential of
32 mV (12, 17, 18). Wet weight of cells was calculated by
multiplying dry mass by 4.77 (1).
Materials. Phentolamine was from Sigma Chemical (St. Louis, MO). BSA was from Boehringer Mannheim Biochemicals (Indianapolis, IN or Lewes, East Sussex, UK). Radiochemicals were from DuPont-NEN Radiochemicals (Boston, MA or Hounslow, Middlesex, UK). All other chemicals were analytical grade from Ajax Chemicals (Auburn, NSW, Australia) or Sigma Chemical (Poole, Dorset, UK).
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RESULTS |
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The contribution of the mitochondrial proton cycle to the respiration
rate of the contracting hindquarter was determined as described by
Rolfe and Brand (24), and the results are shown in Fig.
2. Each individual experiment to determine
the contribution of proton leak (plus nonmitochondrial oxygen
consumption) and ATP turnover to the respiration rate of the
contracting hindquarter lasted ~3 h. Steady-state values for resting
oxygen consumption and average were reached in ~20 and ~90
min, respectively. Hence the resting values for respiration rate and
were measured after 1.5 h of perfusion. Contraction was then
induced in the gastrocnemius-plantaris-soleus group of the left
hindquarter, and the rates of hindquarter oxygen consumption and
average membrane potential were measured after 15 min of contraction
when a new steady state for these parameters had been attained.
Contraction increased the respiration rate of the hindquarter by
~80% on average but did not alter the value for total tissue
membrane potential as measured by the TPMP signal, although, since a
drop in plasma membrane potential of 20 mV was assumed to occur in the
contracting tissue, this meant that the calculated total tissue
mitochondrial
actually increased (by ~4 mV) during
contraction.
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After 45 min of contraction, during which the stability of the steady
state was verified by constant measurement of and oxygen
consumption, sufficient oligomycin to completely inhibit oxidative ATP
synthesis (~50 µg/g perfused tissue) was added, and, 15 min after
this addition, the new steady-state values of
and oxygen
consumption were measured. Oligomycin was shown to be in excess by
demonstrating no effect on respiration of further additions. Note also
that all contraction was abolished 5-10 min after addition of
excess oligomycin. Addition of oligomycin reduced the hindquarter
respiration rate by 51% and increased the
by ~20 mV. As
discussed previously (24), to measure the contribution of proton leak
(plus nonmitochondrial oxygen consumption) to tissue respiration rate,
it is necessary to return the tissue
to its contracting,
pre-oligomycin inhibition value. This was achieved by titrating the
oligomycin-inhibited hindquarter with cyanide as outlined previously
(24). The nonmitochondrial oxygen consumption rate was determined using
excess (1-2 mM) sodium cyanide as described previously (24);
additional cyanide did not decrease respiration further.
Figure 2 shows that the contributions of proton leak, ATP turnover, and nonmitochondrial oxygen consumption to total tissue respiration rate in the contracting state were 34 ± 14, 57 ± 21, and 9 ± 7% (means ± SE), respectively. Thus the proton leak was still a major contributor to respiration rate even in the contracting state.
The contributions of proton leak, ATP turnover, and nonmitochondrial oxygen consumption to respiration in noncontracting muscle can also be calculated from Fig. 2; they were 57, 27, and 16%, respectively, which agrees well with our previous estimates (52, 34, and 14%, respectively; Ref. 24), despite differences in experimental conditions (rat strain, perfusion with red blood cells, temperature 32 compared with 35°C).
Addition of lactate, pyruvate, glutamate, and ornithine to the basal
incubation medium increased hepatocyte respiration from 1.01 ± 0.05 to 2.22 ± 0.33 µmol
O2 · min1 · g
wet weight
1 (means ± SE; data not shown). The contribution of proton leak to the oxygen
consumption rate of isolated liver cells in this supplemented medium is
shown in Fig. 3 and was determined in
essentially the same way as described above for perfused muscle except
that myxothiazol (an inhibitor of complex III) was used instead of cyanide. Figure 3 shows that the respiration rate of these cells was
accounted for by mitochondrial proton leak (22 ± 2%, mean ± SE), ATP turnover (69 ± 2%, mean ± SE), and nonmitochondrial oxygen consumption (9 ± 1%, mean ± SE). Thus proton
leak remains a significant contributor to the respiration rate of
hepatocytes in which ATP turnover has been stimulated to give double
the respiration rate.
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DISCUSSION |
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The data in Fig. 2 indicate that when the muscle is stimulated to
contract the value of the average mitochondrial does not change
and indeed may even increase slightly. This indicates that both the
pathways involved in using the mitochondrial
(ATP turnover) and
those involved in producing the mitochondrial
(by oxidation of
substrates) are activated. The fact that the average value of
mitochondrial
remains constant during the rest-to-exercise
transition means that the rate of proton leak across the mitochondrial
inner membrane (which is controlled by
) also remains constant,
and this explains why the proton leak is still a major contributor to
the respiration rate of the exercising hindquarter. However, perfused
muscle is a complicated system, and, although it is clear that there is
activation of the
producers during contraction, the conclusion
of equal activation of
producers and consumers must be
considered tentative.
In microsphere entrapment studies, it has been shown that, in the
constant-flow-perfused hindlimb, perfusate flow is diverted from the
nonworking to the working tissue during exercise (Newman and Clark,
unpublished data), and such flow changes might lead us to misinterpret
the data shown in Fig. 2. Diversion of flow from the nonworking tissue
may cause the oxygen consumption rate of this part of the hindquarter
to be limited by suboptimal substrate delivery, leading to a drop in
the signal from this part of the hindquarter. However, the
effect of this drop on the total tissue membrane potential signal would
be either to accentuate any
drop or to mask any
increase
in the contracting tissue. The data in Fig. 2 show no change or even an
increase in the total mitochondrial
on induction of exercise and
thus, if anything, may underestimate a hypothetical
increase in
the contracting tissue.
Only 15% of the total muscle mass of the hindquarter was stimulated to
contract, as estimated by dye infusion (10, 16, 28). This means that
any changes in the mitochondrial in this portion of the
hindquarter would have been difficult to detect against the background
of the total tissue
. For example, a change of 40 mV in the
mitochondria within the exercising tissue would only produce a 4-mV
change in the total tissue membrane potential signal. Thus it is
possible that the
of the contracting tissue actually dropped but
that we were not able to detect this drop, weakening our conclusion
that substrate oxidation is activated during exercise induction.
However, even a 40-mV drop in the total tissue mitochondrial
would, from Fig. 2, only decrease the contribution of proton leak to
the respiration rate of contracting hindquarter from 34 to 20%, thus
reducing our estimate of the contribution of proton leak in liver and
skeletal muscle to SMR from 15 to 10% and the total estimated
contribution of leak in all tissues to 15%. Thus the difficulty of
accurately measuring changes in mitochondrial
does not affect
the main conclusion of Fig. 2, that proton leak is a major contributor
to the respiration rate of perfused, contracting skeletal muscle under
conditions that may reflect those of standard metabolism in vivo.
How close are the conditions in our experiments to the standard state
in vivo? As mentioned in the introduction, it is thought that a certain
amount of muscular contraction occurs in standard metabolism (23, 25)
and that the liver may have some gluconeogenic and ureagenic activity.
We have increased by exercise induction the total respiration rate of
the hindquarter to close to the maximum rate of oxygen consumption
measured for resting skeletal muscle in vivo [0.787 µmol
O2 · min1 · g
tissue
1 (21) compared with
0.65 µmol
O2 · min
1 · g
tissue
1 (Fig. 2)],
and from this point of view our contracting hindquarter preparation is
a better model of the standard state than our resting preparation (24).
However, one of the main differences between perfused muscle and real
life is that during SMR in vivo most of the muscle tissue is
contracting a small amount, whereas in our system a small proportion of
the total mass is contracting maximally and the remainder is resting.
Thus the strength of our conclusion rests on the assumption that the
mechanism by which muscle metabolism is stimulated when muscle tissue
is exercising lightly is the same as when it undergoes maximal
contraction. If, for example, only the ATP turnover reactions were
stimulated during light exercise, leading to a drop in the
mitochondrial
, then our model system would overestimate the
contribution of proton leak to standard metabolism in skeletal muscle.
However, as mentioned above, the drop in mitochondrial
would
have to be very large (>60 mV) to significantly affect our main
conclusion, that mitochondrial proton leak is a significant contributor
to SMR.
The experiments with liver cells were designed (as for muscle) to
stimulate ATP-consuming pathways to somewhere near physiological levels. One crude assessment of whether we achieved this can be made by
comparing the respiration rate of whole liver in vivo (2.84 µmol
O2 · min1 · g
liver
1; Ref. 13) with the
respiration rate of our liver cells, scaled in the appropriate way.
Knowing the number of cells per whole liver
(~109; Ref. 1) and the wet
weight per cell (~8 g/109 cells;
Ref. 1), we can calculate that the respiration rate of our
experimental cell population was equivalent to 1.8 µmol O2 · min
1 · g
liver
1 (64% of the in vivo
rate). By this measure, our cell population was representative of liver
cells in vivo.
The results in Figs. 2 and 3 show that the contribution of the
mitochondrial proton cycle to respiration in stimulated skeletal muscle
and liver cells is large, as it is in resting muscle and hepatocytes
(4, 24), although stimulation reduces the absolute magnitude of the
contribution of proton leak from 52 ± 15 to 34 ± 14% (means ± SE; significantly different at the
P < 0.10 level) in muscle and from
26 ± 7 to 22 ± 2% (means ± SE; significantly different at
the P < 0.01 level) in liver cells.
Rolfe and Brand (24) discussed the effect of the contribution of proton
leak on the effective P/O ratio (mol ATP synthesized/total mol atomic oxygen consumed) in intact tissues and calculated that the effective P/O ratio in resting skeletal muscle would be 0.83. The effective P/O
ratio in resting liver has been estimated as ~1.3-1.6 (4). The
effective P/O ratio is obtained by multiplying the mechanistic P/O
ratio of the mitochondria by the fraction of total tissue oxygen
consumption used to drive mitochondrial ATP synthesis. The proportion
of hindquarter oxygen consumption used to drive ATP synthesis was 57%
in our contracting hindquarter system (Fig. 2) but was 34% in resting
skeletal muscle (24). Thus the effective P/O ratio of skeletal muscle
at almost double the resting respiration rate (conditions that may
better reflect SMR) can be calculated, under the assumption that the
muscle is oxidizing pyruvate, for which the mechanistic P/O ratio is
2.42 (2), to be ~1.4 (0.57 × 2.42). The same calculation for
liver, with 69% of liver oxygen consumption used to drive
mitochondrial ATP synthesis (Fig. 3), gives an effective P/O ratio of
~1.7. Thus the data presented in this paper support those of other
workers (e.g., Refs. 5, 6) who consider that
31P-nuclear magnetic resonance
saturation transfer measurements of
Pi ATP flux may
significantly overestimate the effective P/O ratio of intact tissues.
We therefore consider that the conclusion of others (e.g., Refs. 14,
19) that all of the oxygen consumption of intact tissues is used to
drive ATP synthesis is incorrect even when respiration rate is doubled
compared with the resting state (see Ref. 24 for a fuller discussion of
this topic).
The contribution of proton leak, ATP turnover, and nonmitochondrial
oxygen consumption to SMR may be calculated using the results shown in
Figs. 2 and 3. The contributions of liver and skeletal muscle to rat
SMR are 10-20 and 13-42%, respectively (see references cited
in Ref. 24). Hence, calculation using the values for the contribution
of proton leak to liver and skeletal muscle respiration [22%
(Fig. 3) and 34% (Fig. 2), respectively] indicates that the
contribution of liver and skeletal muscle proton leak to rat SMR is
between 7% [(10% × 0.22) + (13% × 0.34)] and 19% [(20% × 0.22) + (42% × 0.34)], with an
average of ~15%. If the contribution of proton leak in the other
major oxygen-consuming tissues of the rat (which together account for
20% of rat SMR; Ref. 10) is similar to liver, then the contribution of
proton leak to rat SMR may be as high as 23% [(20% × 0.22) + (42% × 0.34) + (20% × 0.22)]. With the same
assumptions and the value of 9% (see Figs. 2 and 3) obtained for the
contribution of nonmitochondrial oxygen consumption to the respiration
rate of liver and skeletal muscle, the contribution of nonmitochondrial
oxygen consumption to rat SMR could be as high as 7% [(20% × 0.09) + (42% × 0.09) + (20% × 0.09)]. Total
ATP turnover, which accounts for 57% of skeletal muscle respiration
(Fig. 2) and 69% of liver cell respiration (Fig. 3), would contribute
a maximum of 52% [(20% × 0.69) + (42% × 0.57) + (20% × 0.69)] to rat SMR. The significance of the
contribution of proton leak and nonmitochondrial oxygen consumption in
liver and skeletal muscle to rat SMR is shown in Fig.
4 and compared with available data for
protein synthesis and
Na+-K+-ATPase
and Ca2+-ATPase activity in the
rat, these being the only processes for which there is general
agreement regarding their contribution to mammalian SMR.
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What are the confidence limits of our estimate of the contribution of proton leak to SMR? These limits are difficult to quantify precisely, but the following points should be noted. First, we have (by stimulating the muscle ATP demand via contraction) pushed the oxygen consumption rate of the hindquarter close to the upper limit of estimates for the resting in vivo oxygen consumption rate of the rat hindlimb (see above), and this would, if anything, tend to underestimate the contribution of proton leak to skeletal muscle respiration. Second, we have not used free fatty acids as a substrate in this study, despite the fact that muscle may use lipids as the main fuel source for SMR. Many studies have shown that free fatty acids can uncouple mitochondria by increasing the activity of the proton cycle (e.g., Ref. 7), and so the use of pyruvate and glucose as the fuel source in this study would tend to underestimate the proton leak activity in muscle. These points indicate that the value for the contribution of proton leak to SMR derived from the data contained in this paper represents a lower-limit estimate.
In what ways could we have overestimated the contribution of proton
leak to SMR? First, the estimate for nonmitochondrial oxygen
consumption in the contracting tissue may have been misestimated, as
contraction is necessarily abolished when cyanide is added to allow
measurement of nonmitochondrial oxygen consumption rate. However, it is
not clear whether this would lead to an overestimate or underestimate
of nonmitochondrial oxygen consumption (and therefore of proton leak
activity), or indeed whether nonmitochondrial oxygen consumption would
be affected at all by muscle contraction. Second, the release of
Ca2+ from the sarcoplasmic
reticulum (SR) as a result of sciatic nerve stimulation could cause
mitochondrial uncoupling (and therefore an overestimate of proton leak
activity) because mitochondrial ATP synthesis, which would normally
drive the reuptake of Ca2+ by SR
Ca2+-ATPases, was abolished in our
experiments (by addition of oligomycin) before the proton cycle
activity was measured. However, as mentioned in
RESULTS, the contribution of proton
leak to the respiration rate of noncontracting muscle calculated from
the data in Fig. 2 is essentially the same as in our previous paper
(Ref. 24, in which Ca2+ release
from the SR was blocked by dantrolene). In addition, preliminary
experiments using dantrolene as described previously (24) showed that
the proton leak activity in the presence of dantrolene-oligomycin (0.29 ± 0.07 µmol
O2 · min1 · g
tissue
1; mean ± SE,
n = 2) was not significantly different
from that of subsequent experiments (0.33 ± 0.11 µmol
O2 · min
1 · g
tissue
1; mean ± SE,
n = 5). Dantrolene was not used in the
latter set of experiments because, in contrast to our previous work,
the preliminary experiments showed that addition of oligomycin did not
cause a drop in the average tissue membrane potential, a drop that
dantrolene had previously been used to prevent (24). Note that, in the
same preliminary experiments, tissue pH gradients were measured and
were found to be unaffected by the addition of oligomycin after
contraction was initiated (data not shown), in agreement with our
previous studies. However, the plasma membrane potential was not
monitored in the preliminary experiments (or in the subsequent ones),
and this omission might have caused us to misestimate the proton leak
activity; nevertheless, we have shown that oligomycin and cyanide have
no effect on the plasma membrane potential in noncontracting muscle
(24), and we feel that it is unlikely that this would be different in
contracting muscle. Finally, the development of edema, which occurs
toward the end of long perfusions, may artificially increase the proton leak activity. However, we have previously shown (24) that the rate of
respiration of the perfused tissue in the presence of saturating
amounts of oligomycin is constant despite development of edema in the
hindlimbs similar to that seen in the experiments described in this
paper. In general, therefore, we consider that the data in this paper
represent a reasonably accurate underestimate of the contribution of
proton leak to the oxygen consumption rate of lightly working skeletal muscle.
In conclusion, we have shown that proton leak accounts for ~34 and ~22% of the resting oxygen consumption rates in perfused skeletal muscle and isolated liver cells, respectively, under conditions in which respiration rate is double the resting value, which may be a reasonable approximation to the maximal resting rates of liver and skeletal muscle in vivo. However, in the case of the muscle preparation, although the whole preparation is more than doubling its respiration rate, that of the contracting tissue, which represents 15% of the whole preparation, is increasing >10-fold, and thus any extrapolation to the in vivo condition should be taken with caution. We have shown that proton leak in skeletal muscle and liver at double their resting respiration rates accounts for between 7 and 19% (mean 15%) of rat SMR. If proton leak activity is similar in other tissues to that seen in liver, the contribution of proton leak to rat SMR would be between 11 and 23% (mean 19%). We conclude that proton leak is a major contributor to rat SMR even when the respiration rates of tissues in the standard state are significantly above their resting rates.
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ACKNOWLEDGEMENTS |
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We thank Simon Price for his contribution to the work with hepatocytes.
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FOOTNOTES |
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This work was supported by grants from the Royal Society (to D. F. S. Rolfe), the Australian Research Council (to M. G. Clark), and the Biotechnology and Biological Sciences Research Council (to M. D. Brand).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and present address of D. F. S. Rolfe: Dept. of Biomedical Science, University of Wollongong, Northfields Ave., Wollongong 2522, NSW, Australia.
Received 2 June 1998; accepted in final form 20 November 1998.
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REFERENCES |
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1.
Berry, M. N.,
C. Farrington,
S. Gay,
A. R. Grivell,
and
P. G. Wallace.
Characterisation of the isolated hepatocyte preparation.
In: Isolation, Characterisation and Use of Hepatocytes, edited by R. A. Harris,
and W. Neal. New York: Elsevier Science, 1983, p. 50-54.
2.
Brand, M. D.
The stoichiometry of proton pumping and ATP synthesis in mitochondria.
Biochemist
16:
20-24,
1994.
3.
Brand, M. D.
Measurement of mitochondrial protonmotive force.
In: Bioenergetics: A Practical Approach, edited by G. C. Brown,
and C. E. Cooper. Oxford, UK: IRL, 1995, chapt. 3, p. 39-62
4.
Brand, M. D.,
L.-F. Chien,
E. K. Ainscow,
D. F. S. Rolfe,
and
R. K. Porter.
The causes and functions of mitochondrial proton leak.
Biochim. Biophys. Acta
1187:
132-139,
1994[Medline].
5.
Brindle, K. M.,
M. J. Blackledge,
R. A. J. Chaliss,
and
G. K. Radda.
31P NMR magnetization-transfer measurements of ATP turnover during steady state isometric muscle contraction in the rat hind limb in vivo.
Biochemistry
28:
4887-4893,
1989[Medline].
6.
Brindle, K. M.,
and
G. K. Radda.
31P-NMR saturation transfer measurements of exchange between Pi and ATP in the reactions catalysed by glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase in vitro.
Biochim. Biophys. Acta
928:
45-55,
1987[Medline].
7.
Brown, G. C.,
and
M. D. Brand.
On the nature of mitochondrial proton leak.
Biochim. Biophys. Acta
1059:
55-62,
1991[Medline].
8.
Clausen, T.,
C. van Hardeveld,
and
M. E. Everts.
Significance of cation transport in control of energy metabolism and thermogenesis.
Physiol. Rev.
71:
733-774,
1991
9.
Colquhoun, E. Q.,
M. Hettiarachchi,
J.-M. Ye,
E. A. Richter,
A. J. Hniat,
S. Rattigan,
and
M. G. Clark.
Vasopressin and angiotensin II stimulate oxygen uptake in the perfused rat hindquarter.
Life Sci.
43:
1747-1754,
1988[Medline].
10.
Dora, K. A.,
S. Rattigan,
E. Q. Colquhoun,
and
M. G. Clark.
Aerobic muscle contraction impaired by serotonin-mediated vasoconstriction.
J. Appl. Physiol.
77:
277-284,
1994
11.
Field, J.,
H. S. Belding,
and
A. W. Martin.
An analysis of the relation between basal metabolism and summated tissue respiration in the rat.
J. Cell. Comp. Physiol.
14:
143-155,
1939.
12.
Harper, M.-E.,
and
M. D. Brand.
The quantitative contributions of mitochondrial proton leak and ATP turnover reactions to the changed respiration rates of hepatocytes from rats of different thyroid status.
J. Biol. Chem.
268:
14850-14860,
1993
13.
Jansky, L.
Adaptability of heat production mechanisms in homeotherms.
Acta Univ. Carol. [Med.] (Praha)
1:
1-91,
1965.
14.
Kingsley-Hickman, P. B.,
E. Y. Sako,
P. Mohanakrishnan,
P. M. L. Robitaille,
A. H. L. From,
J. E. Foker,
and
K. Ugurbil.
31P NMR studies of ATP synthesis and hydrolysis kinetics in the intact myocardium.
Biochemistry
26:
7501-7510,
1987[Medline].
15.
Lannergren, J.,
and
H. Westerblad.
Fatigue mechanisms in single Xenopus muscle fibres of different types.
In: Muscle Energetics, edited by R. J. Paul,
G. Elzinga,
and K. Yamada. New York: Liss, 1989, p. 99-107.
16.
McAllister, R. M.,
and
R. L. Terjung.
Training-induced muscle adaptations: increased performance and oxygen consumption.
J. Appl. Physiol.
70:
1569-1574,
1991
17.
Nobes, C. D.,
G. C. Brown,
P. N. Olive,
and
M. D. Brand.
Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes.
J. Biol. Chem.
265:
12903-12909,
1990
18.
Porter, R. K.,
and
M. D. Brand.
Causes of the differences in respiration rate of hepatocytes from mammals of different body mass.
Am. J. Physiol.
269 (Regulatory Integrative Comp. Physiol. 38):
R1213-R1224,
1995
19.
Radda, G. K.
Control, bioenergetics and adaptation in health and disease: noninvasive biochemistry from nuclear magnetic resonance.
FASEB J.
6:
3032-3038,
1992
20.
Rattigan, S.,
K. A. Dora,
A. C. Y. Tong,
and
M. G. Clark.
Perfused skeletal muscle contraction and metabolism improved by angiotensin II-mediated vasoconstriction.
Am. J. Physiol.
271 (Endocrinol. Metab. 34):
E96-E103,
1996
21.
Rennie, M. J.,
and
J. O. Holloszy.
Inhibition of glucose uptake and glycogenolysis by availability of oleate in well oxygenated perfused skeletal muscle.
Biochem. J.
168:
161-170,
1977[Medline].
22.
Richter, E. A.,
P. J. F. Cleland,
S. Rattigan,
and
M. G. Clark.
Contraction-associated translocation of protein kinase C in rat skeletal muscle.
FEBS Lett.
217:
232-236,
1987[Medline].
23.
Rohracher, H.
Permanente rhythmische mikrobewegungen des warmbluter-organismus (microvibration).
Naturwissenschaften
49:
145-147,
1962.
24.
Rolfe, D. F. S.,
and
M. D. Brand.
Contribution of mitochondrial proton leak to skeletal muscle respiration and to standard metabolic rate.
Am. J. Physiol.
271 (Cell Physiol. 40):
C1380-C1389,
1996
25.
Rolfe, D. F. S.,
and
G. C. Brown.
Cellular energy utilization and molecular origin of standard metabolic rate in mammals.
Physiol. Rev.
77:
731-758,
1997
26.
Ruderman, N. B.,
C. R. S. Houghton,
and
R. Hems.
Evaluation of the isolated perfused rat hindquarter for the study of muscle metabolism.
Biochem. J.
124:
639-651,
1971[Medline].
27.
Seglen, P. O.
Preparation of isolated rat liver cells.
Methods Cell Biol.
13:
29-83,
1976[Medline].
28.
Spriet, L. C.,
M. I. Lindinger,
G. J. F. Heigenhauser,
and
N. L. Jones.
Effects of alkalosis on skeletal muscle and performance during exercise.
Am. J. Physiol.
251 (Regulatory Integrative Comp. Physiol. 20):
R833-R839,
1986[Medline].
29.
Waterlow, J. C.
Protein turnover with special reference to man.
Q. J. Exp. Physiol.
69:
409-438,
1984[Medline].