Age-related increase in mitochondrial proton leak and decrease
in ATP turnover reactions in mouse hepatocytes
Mary-Ellen
Harper1,
Shadi
Monemdjou1,
Jon J.
Ramsey2, and
Richard
Weindruch2,3
1 Department of Biochemistry,
Microbiology and Immunology, Faculty of Medicine, University of
Ottawa, Ottawa, Ontario, Canada K1H 8M5;
2 Wisconsin Regional Primate
Research Center, University of Wisconsin, Madison 53715;
and 3 Department of Medicine,
University of Wisconsin, and Veterans Administration Geriatric
Research, Education and Clinical Center, Madison, Wisconsin 53705
 |
ABSTRACT |
Age-related changes in mitochondria, including
decreased respiratory control ratios and altered mitochondrial inner
membrane lipid composition, led us to study oxidative phosphorylation
in hepatocytes from old (30 mo) and young (3 mo) male C57BL/J mice. Top-down metabolic control analysis and its extension, elasticity analysis, were used to identify changes in the control and regulation of the three blocks of reactions constituting the oxidative
phosphorylation system: substrate oxidation, mitochondrial proton leak,
and the ATP turnover reactions. Resting oxygen consumption of cells
from old mice was 15% lower (P < 0.05) than in young cells. This is explained entirely by a decrease in
oxygen consumption supporting ATP turnover reactions. At all values of
mitochondrial membrane potential assessed, the proportion of total
oxygen consumption used to balance the leak was greater in the old
cells than in the young cells. Metabolic control coefficients indicate
a shift in control over respiration and phosphorylation away from
substrate oxidation toward increased control by leak and by ATP
turnover reactions. Control of the actual number of ATP molecules
synthesized by mitochondria for each oxygen atom consumed by the ATP
turnover and leak reactions was greater in old than in young cells,
showing that efficiency in older cells is more sensitive to changes in these two blocks of reactions than in young cells.
oxidative phosphorylation; uncoupling; oxidative stress; free
radicals; aging
 |
INTRODUCTION |
THE CONCEPT THAT FREE RADICALS are involved in key
processes resulting in aging was first proposed over 40 years ago (17, 23). In recent years evidence has corroborated and extended the
original free radical theory of aging, and together the evidence has
been amalgamated into the now widely supported oxidative stress hypothesis of aging (1, 41, 42). The latter implicates endogenous
oxidants in senescence-related loss of cellular function. The
underlying tenet of the original free radical theory was that free
radicals caused the progressive and irreversible damage and deterioration of cellular function. In contrast to the free radical theory, the oxidative stress hypothesis of aging does not limit the
source of the damaging metabolites to oxygen-derived free radicals;
moreover, the scope has been expanded from oxidant-induced molecular
damage to include oxidant modulation of gene expression, signal
transduction, and other normal processes (40, 41, 46, 48).
Despite over 40 years of research, there is a distinct lack of
integrative data on the effects of oxidative stress on the metabolism
of intact cells. Gerontological investigations of mitochondrial function have been limited almost exclusively to isolated mitochondria. Mitochondrial function, as assessed by analyses of isolated
mitochondria and mitochondrial enzymes, is affected by aging (22, 29,
40). Mitochondrial components damaged through oxidative mechanisms include mitochondrial DNA (which encodes for several proteins of the
electron transport chain), proteins, and lipids. Compared with nuclear
DNA, the extent of oxidative damage to mitochondrial DNA
(mtDNA) is much greater. Damage to mtDNA from rat liver or human brain
regions is at least tenfold more than that in nuclear DNA (33, 39).
There are a number of reasons explaining the vulnerability of mtDNA to
oxidative damage. Beyond the fact that mitochondria are responsible for
the production of the majority of free radicals in cells, the DNA is
immediately juxtaposed to the electron transport chain in the
mitochondrial inner membrane (MIM), where the oxidants are produced.
Moreover, mtDNA lacks the protective histones and extensive DNA repair
capabilities that protect nuclear DNA. The function of many
mitochondrial proteins is impaired with age. For example, the
activities of complexes I, II, and IV decrease with age in postmitotic
tissues (3, 5, 14, 34, 43).
Effects of aging on MIM lipids include increased levels of long-chain
polyunsaturated fatty acids such as 22:4 and 22:5 and decreases in
18:2, 18:1, and 16:1. As a result, with increases in age there is an
increased probability of membrane lipid peroxidation (the
"peroxidizability index") (30, 49). Linoleic acid (18:2) is
thought to be necessary for the optimal interactions of cardiolipin with many substrate transporters and components of the MIM (28, 29).
Shigenaga et al. (40) hypothesized that these decreases in inner
membrane lipids containing 18:2 and altered cardiolipin-protein interactions may account for the decreased State 3-to-State 4 ratio
(State 3/State 4) and contribute to the loss of efficiency in
mitochondrial function. Our results support and extend this line of
thought.
Although isolated mitochondria have been extensively investigated in
aging studies, it is known that populations of mitochondria from
tissues of old and young organisms can be differentially damaged by
mitochondrial isolation procedures. Histological studies have shown
that, with increasing age, mitochondria tend to be larger, there is
increased matrix vacuolization, cristae are shorter, and there is a
decrease in dense granules (47). Importantly, it has been estimated
that only about one-half of these enlarged mitochondria are retained in
mitochondrial isolations from tissues of older animals (47). Thus the
quality of the mitochondrial preparation from old organisms may, in
fact, be improved by the loss of unhealthy mitochondria during the
isolation procedures (47), leading potentially to artifactual
conclusions about changes in mitochondrial function with age. Moreover,
oxidative damage occurs throughout the cell, emphasizing the importance
of metabolic studies conducted using intact cells and tissues.
The experimental approach that we have used is one that has been useful
in identifying the sites of action of hormones and other external
effectors in metabolic pathways in intact cells as well as in
mitochondrial preparations. The approach is referred to as top-down
elasticity analysis (7), and it is an extension of metabolic control
analysis. Whereas metabolic control analysis allows the identification
of the important sites of control within metabolic pathways, top-down
elasticity analysis is used in the comparison of varying metabolic
conditions to identify differences in pathway regulation. The latter is
extremely useful, for example, in the identification of the "sites
of action" of hormones, drugs, and metabolic defects. Some of the
useful measurements to emerge from an elasticity analysis include
"elasticity coefficients," "flux control coefficients," and
"concentration control coefficients." In very general terms, an
elasticity coefficient (often referred to simply as an
"elasticity") describes the responsiveness of a branch of a
metabolic pathway to changes in the amount of an intermediate in that
pathway. If the elasticity for a branch differs between the
drug-treated and the control pathways being compared, then one site of
action of the drug is located within the reactions encompassed by that
branch. Other measurements include flux control and concentration
control coefficients. Values of the latter describe, respectively, the
relative proportion of control by branches of the pathway over the rate
of the pathway and over the amounts of intermediates. Several useful
reviews on this approach have been published recently (see Refs. 8,
26). In intact cells it has been successfully used to investigate the
sites of action of glucagon (9), thyroid hormones (19, 20, 24, 25), and
butylated hydroxyanisole (16) in mitochondria, and of fatty acids in
isolated hepatocytes of rats (37). Here we use the approach to
quantitatively identify in mouse hepatocytes the effects of aging on
reactions that are central to oxidative phosphorylation.
On the basis of the above documented age-related changes in
mitochondria, particularly the decreases in the State 3/State 4 and
changes in MIM lipid composition, we hypothesized that the mitochondrial proton leak might be greater in cells from old compared with young mice. Thus we aimed, in the following set of experiments, to
compare the overall kinetics of the mitochondrial proton leak in
hepatocytes from old (30 mo) and young (3 mo) mice. We also conducted
the first complete metabolic control analysis on oxidative phosphorylation in intact cells from old mice and report herein that
the overall kinetics of the mitochondrial proton leak are altered by
aging. Moreover, we show that there is a significant decrease in the
amount of oxygen used to support the synthesis and use of ATP in old
compared with young cells. Top-down metabolic control analysis showed
that there is a shift in control away from substrate oxidation
reactions toward increased control by the leak and by ATP turnover
reactions in hepatocytes from old mice.
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EXPERIMENTAL PROCEDURES |
Treatment of animals.
Twenty-seven male 30-mo-old C57BL/6J mice were received from the
Veterans Administration Geriatric Research, Education, and Clinical
Center at the University of Wisconsin (Madison, WI). These mice were
part of a cohort purchased at 1 mo of age from Charles River
Laboratories (Wilmington, DE), group housed (three per cage), and given
free access to Purina 5001 chow diet and water. Twenty-seven young (3 mo) control C57BL/6J mice were obtained from The Jackson Laboratory
(Bar Harbor, ME). On receipt in Ottawa, mice were caged individually at
23°C with light from 0700 to 1900 and were given free access to a
Purina 5001 chow diet and water. For the isolation of hepatocytes,
nonfasted mice were anesthetized with 1 mg of pentobarbitone/100 g body
weight and were killed between 0730 and 1100. Mice used in this study
were cared for in accordance with the principles and guidelines of the
Canadian Council on Animal Care, the Institute of Laboratory Animal
Resources (National Research Council, USA), and with the
Guiding Principles for Research Involving Animals and
Human Beings.
Isolation and incubation of hepatocytes.
Hepatocytes were isolated and incubated as earlier described (19). The
viability of cells was >92% as determined by the exclusion of 0.3%
(wt/vol) trypan blue. Before incubations, the cells were stored on ice
in the isolation medium containing (in mM) 148 NaCl, 5 KCl, 0.81 MgSO4 · 7H2O,
0.83 Na2HPO4,
0.14 KH2PO4, 1 CaCl2, 25 NaHCO3, and 15 glucose.
For incubations, the cells were diluted approximately sevenfold in an
incubation medium containing (in mM) 106 NaCl, 5 KCl, 25 NaHCO3, 0.41 MgSO4, 10 Na2HPO4,
2.5 CaCl2, 10 glucose, 10 lactate, 1 pyruvate, and 2.25% (wt/vol) defatted BSA. Stock 9% BSA was defatted by the method of Chen (12) and dialyzed against 153 mM NaCl
and 11 mM KCl. Cell suspensions (3-5 ml of 6-9 mg dry wt
cells/ml) were incubated in 20-ml stoppered glass vials at 37°C in
a shaking water bath (100 cycles/min). To allow equilibration of the
medium to a pH of 7.4, the gas phase above each suspension during
incubations was 95% air-5% CO2.
The cells were preincubated at 37°C in the shaking water bath for
10 min to allow the hepatocytes to reestablish ion gradients after
being stored on ice. Cells were then incubated a further 20 min in the
presence of the various inhibitors, uncouplers, and isotopes before
aliquots were taken for the measurements of oxygen consumption and
mitochondrial membrane potential
(
m). (Refer to
Application of top-down elasticity analysis and
top-down control analysis and to legends of Figs. 1-4 for the specific concentrations of inhibitors and uncouplers used in each experiment.)
Measurement of oxygen consumption.
The respiration rate of hepatocytes was measured using a Hansatech
(Norfolk, UK) Clark-type oxygen electrode, the incubation chamber of
which was maintained at 37°C and magnetically stirred. Each rate
was assessed in duplicate using 1.0 ml of cell suspension. Respiration
rates are reported here as per 106
cells rather than per milligram dry weight of cells, as is more normal
for rat hepatocyte preparations. This is simply because the yield of
hepatocytes from a mouse is about one-tenth of that from a rat, and an
inordinate amount of the final cell preparation would be needed to
accurately assess dry cell weight. Cell counts were determined with a
hemocytometer. All respiration rates were determined simultaneously and
in parallel with the measurements of

m. The resting respiration
rate was defined as the oxygen consumption rate in the absence of
inhibitors and uncouplers. Nonmitochondrial oxygen consumption was
determined after the incubation of cells with maximal concentrations of
oligomycin (1 µg/ml) and antimycin (5 µM) and with valinomycin (0.1 µM) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone
(FCCP) (20 µM).
Measurement of

m.
Total aqueous volume, extracellular volume, and

m were estimated from the
distributions of
3H2O,
[14C]methoxyinulin,
and
[3H]methyltriphenylphosphonium
cation (TPMP+) as described
previously (25, 36). 
m can
be calculated by knowing the proportion of cytoplasmic volume that is
occupied by the mitochondrial matrix [mitochondrial volume/cell
volume (Vm/Vc)],
the apparent activity coefficient of
TPMP+ in each compartment
(ae,
ac, and
am; where
subscripts e, c, and m represent extracellular, cytoplasmic plus
nuclear, and mitochondrial), and the extent of the accumulation of
TPMP+ into the whole cell
([TPMP+]t/[TPMP+]e)
and into the cytoplasm in relation to the external medium ([Cl
]e/[Cl
]t).
The relationship between 
m
and TPMP+ distribution at 37°C
is shown below
The
determination and the actual values of these correction factors are
outlined briefly in the following paragraphs.
Vc and
Vm values were determined using
quantitative morphometric cytology (45) as described previously (25).
Briefly, isolated cells were fixed with glutaraldehyde, and electron
micrographs of osmium-stained cells were prepared at a final
magnification of 4,950-fold. Mitochondrial volume was determined from
the number of intersections of a 1-cm grid overlaying the micrographs.
Then the volume was calculated as the total number of intersections in
mitochondria divided by the total number of intersections in cells
(less the total number of intersections in lipid droplets). The
cellular volume was corrected for the volume of lipid droplets, because
TPMP+ is not taken up into fat
(13). Mitochondrial matrix volume was calculated as 56.5% of total
mitochondrial volume on the basis of the work of Loud (32), who
calculated this weighted mean from the percentage matrix volume of
midzonal, peripheral, and central liver cells and the percentage of
these cell types in the whole liver.
[Cl
]e/[Cl
]t
and plasma membrane potential
(
p) were determined from
the distribution of 36Cl (35).

p was 36.0 mV ± 6.2 (n = 3) and 39.3 mV ± 0.7 (n = 3) in cells from old and young
mice, respectively.
The proportion of TPMP+ that is
free (i.e., not bound) in mitochondria
(am) and the
cytoplasm (ac)
was determined as described by Nobes et al. (36). Values for
am were 0.247 (± 0.039; n = 2) and 0.171 (± 0.073; n = 3) for old and young,
respectively. The
ac values were
0.356 (± 0.019; n = 2) and 0.322 (± 0.015; n = 2) for old and young,
respectively.
At the end of each incubation, triplicate aliquots (0.70 ml) were
removed and pipetted into 1.5-ml minitubes and immediately centrifuged
in a minicentrifuge for 2 min. Then 200-µl aliquots of the
supernatant were removed and pipetted into scintillation vials and
immediately mixed with scintillant. The residual supernatant was
aspirated; the sides of each tube were wiped dry, and 40 µl of 20%
(vol/vol) Triton X-100 were added. After the suspension of the pellet
by vortex mixing, the bottom of the tube was cut off into a
scintillation vial and the pellet was resuspended in 3.0 ml of
scintillant. The radioactivities of the supernatant and pellet were
determined by dual-channel scintillation counting for
3H and
14C by use of the appropriate
quench and crossover corrections.
The apparent volume of pellet available to each isotope (its space in
µl) was calculated as disintegrations per minute in total pellet
divided by disintegrations per minute of supernatant sample. The
[3H]TPMP+
accumulation ratio,
([TPMP+]t/[TPMP+]e),
was calculated as
([3H]TPMP+
space
[14C]methoxyinulin
space)/(3H2O
space
[14C]methoxyinulin
space).
Application of top-down elasticity analysis and top-down control
analysis.
To quantitatively determine the important sites of effects of aging on
oxidative phosphorylation processes, we used the top-down elasticity
analytic approach described by Brand (7). We defined the oxidative
phosphorylation system as the tripartite system shown in Fig.
1 and then determined the overall
elasticities to changes in 
m
of the reactions that produce

m (cellular catabolic reactions, the citric acid cycle, and the electron transport chain) and
those that consume it (ATP synthesis and consumption and the proton
leak). The kinetic response (or elasticity) of the

m producers to

m was measured by titrating
the 
m consumers with oligomycin (0.01-0.05 µg/ml). The kinetic response of the leak to 
m was assessed by
titrating with antimycin (0.05-0.25 µM), an inhibitor of complex
III of the respiratory chain, in the presence of saturating amounts of
oligomycin (1.0 µg/ml). The elasticity of the phosphorylating
subsystem to 
m was measured
from titrations with antimycin alone (0.10-0.20 µM). However,
because the latter titrations provide the kinetics of both

m-consuming subsystems (i.e., the phosphorylating and leak subsystems), corrections were made
for the amount of oxygen required to balance the rate of the proton
leak at each 
m measured.
This was done using the proton leak titration curve.

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Fig. 1.
The oxidative phosphorylation system in hepatocytes. The intermediate
within the system, the mitochondrial membrane potential
( m),
is produced by the substrate oxidation subsystem, which comprises all
of the reactions including and following the oxidation of glucose,
lactate, pyruvate, and endogenous substrates.
 m is consumed by the proton
leak and phosphorylating subsystems. The proton leak subsystem consists
of the leak of protons and any cation cycles across the mitochondrial
inner membrane. The phosphorylating subsystem includes
 m-dependent ATP synthesis
and all cellular ATP-consuming reactions.
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To determine the distribution of control over respiration rate and over

m in cells from old and
young mice, we used top-down control analysis and the published
equations for elasticities, flux control coefficients, and
concentration control coefficients (11, 18).
Statistical analysis.
Data were analyzed using unpaired Student's
t-tests or ANOVA, which was followed
by Tukey's post hoc tests. Linear regression lines were compared by
analysis of covariance with use of Prism 2 for Windows. A
P value of <0.05 was considered
statistically significant. Unless otherwise stated, results are
presented as means ± SE.
Materials.
Oligomycin, antimycin, valinomycin, BSA (fraction V), collagenase (type
IV), inulin, and trypan blue were from Sigma Chemical. FCCP and TPMP
bromide were from Aldrich.
3H2O,
Na[36Cl],
[86Rb]Cl,
[14C]methoxyinulin,
and [3H]TPMP bromide
were from Du Pont NEN. Water-insoluble compounds were dissolved in
dimethyl sulfoxide.
 |
RESULTS |
Resting respiration rates and

m values in
hepatocytes from old and young mice.
The resting respiration rate of hepatocytes from old mice was
significantly less than that of hepatocytes from young controls (P < 0.04). Values were 70.9 ± 3.4 (n = 10) and 83.8 ± 5.1 (n = 7) nmol
O2 · min
1 · 106
cells
1, respectively.
Resting state 
m was not
significantly different between cells from old and young mice. Values
were 149 ± 4.4 mV (n = 8) and 147 ± 3.4 mV (n = 7) in cells
from old and young mice, respectively. State 4 
m values were 155 ± 5.0 mV
(n = 8) and 154 ± 3.0 mV (n = 7), respectively.
Respiration rates and 
m
values are indicated in Fig.
2. The

m results indicate that any
age-induced changes in the 
m
consumers (i.e., leak and ATP turnover in the resting state, and leak
alone in State 4) are matched by any changes in the

m producers (i.e., substrate
oxidation reactions).

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Fig. 2.
Kinetic responses of the mitochondrial proton leak
(A), substrate oxidation
(B), and phosphorylating
(C) subsystems to
 m in hepatocytes from old
and young rats. Oxygen consumption rates are corrected for
nonmitochondrial oxygen consumption. Open symbols, old cells; filled
symbols, young cells. Each symbol marked with an asterisk in
B and
C represents a resting point
[i.e., in the absence of carbonylcyanide
p-trifluoromethoxyphenylhydrazone
(FCCP) and oligomycin]. A:
kinetic response of mitochondrial proton leak to
 m (antimycin titration of
oligomycin-inhibited respiration). B:
kinetic response of substrate oxidation subsystem to
 m. Lines were fitted by
linear regression to oligomycin titration points, showing no difference
in kinetics (P > 0.05 by analysis of
covariance) of substrate oxidation subsystem.
C: kinetic response of the
phosphorylating subsystem to
 m (antimycin titration of
resting respiration rate from which rate of oxygen consumption used to
balance the proton leak has been subtracted). Each point represents
mean ± SE. Results are from cell preparations from the following nos.
of old and young mice, respectively: 8 and 6 (A), 8 and 7 (B), and 5 and 5 (C). Each
 m determination was carried
out in triplicate; each oxygen consumption determination was carried
out simultaneously in duplicate.
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Comparison of the kinetic responses of the mitochondrial proton
leak, substrate oxidation, and phosphorylation subsystems to

m in
hepatocytes from old and young mice.
The kinetic responses of the mitochondrial proton leak to

m in hepatocytes are shown
in Fig. 2A. The oxygen consumption rates are corrected for nonmitochondrial oxygen consumption, as described in EXPERIMENTAL PROCEDURES.
These results show that, over a wide range of

m values, the amount of
oxygen used to support the mitochondrial proton leak is greater in
cells from old mice. Because the kinetics of the proton leak subsystem
are nonlinear, an analysis of covariance could not be used to test for
statistically significant differences. However, taken together, the
results show that the overall kinetics of the mitochondrial proton leak
are altered in hepatocytes from old mice in relation to the results
from young mice.
The kinetic responses of the substrate oxidation subsystem to

m in old and young
hepatocytes are compared in Fig. 2B.
The results indicate that, at any value of

m, there is no difference in
the rate of the 
m-producing
reactions between old and young hepatocytes. Thus there are no
age-related differences in the overall kinetics of the substrate
oxidation reactions.
A comparison of the kinetic responses of the phosphorylating subsystem
to 
m in old and young
hepatocytes revealed marked differences
(P < 0.05; analysis of covariance)
(Fig. 2C). At the resting state, the
rate of the phosphorylating subsystem was ~30% lower in the old
cells than in the young cells at identical values of

m (147 mV).
Quantitative analysis of the effects of the altered kinetics of the
mitochondrial proton leak and phosphorylating subsystems to

m on
respiration rate in old hepatocytes.
The titrations of cellular respiration rate in old and young
hepatocytes that were used to determine the kinetics of the subsystems described in Fig. 2 can be used to quantify the oxygen consumption that
is used to sustain blocks of energy-dissipating reactions: nonmitochondrial oxygen consumption, proton leak reactions, and ATP
turnover reactions as described by Brand (7). The proportion of resting
cellular oxygen consumption that is nonmitochondrial was identified as
that which was insensitive to saturating amounts of antimycin,
oligomycin, valinomycin, and FCCP. There was no significant difference
in the amounts of nonmitochondrial oxygen consumption; values for old
and young cells were 22.1 ± 3.7 (n = 8) and 21.5 ± 1.2 (n = 7) nmol
O2 · min
1 · 106
cells
1, respectively. The
total mitochondrial oxygen consumption and the amounts used to balance
the proton leak and ATP turnover reactions at the resting value of

m are shown in Fig.
3. Despite the fact that the proportion of
resting mitochondrial respiration used to balance the mitochondrial
proton leak is doubled in the cells from old mice compared with young,
there is a small but significant decrease in respiration
(P < 0.04). This decrease can thus
be accounted for entirely by a decrease
(P < 0.03) in the rate of the only
other block of reactions responsible for the dissipation of

m, i.e., ATP synthesis and
consumption reactions.

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Fig. 3.
Proportions of resting mitochondrial oxygen consumption due to proton
leak and ATP turnover reactions. Total oxygen consumption is the
resting rate of mitochondrial oxygen consumption measured in the
absence of any inhibitors or uncouplers and represents the sum of the
oxygen required to fuel the phosphorylation and leak pathways. Total
oxygen consumption is significantly lower in the old (open bars) than
in the young (solid bars, *P < 0.05). Oxygen used to balance mitochondrial proton leak is determined
by extrapolation from titration curves shown in Fig.
2A; oxygen consumption attributable to
leak at resting  m is
subtracted from total resting mitochondrial oxygen consumption.
Statistical analyses show that the apparently greater leak in old cells
does not reach statistical significance. Data were analyzed using ANOVA
and Tukey's post hoc tests. The SE (for total mitochondrial oxygen
consumption) and pooled SE (for proton leak-dependent and ATP
turnover-dependent oxygen consumption) are indicated; the SE for proton
leak at resting  m was
estimated as the mean of the SE for the 2 data points adjacent to it on
proton leak curve in Fig.
2A.
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Application of top-down control analysis.
As well as being useful in the identification of the sites of action of
an external effector and in the quantitative determination of the
importance of changes induced within the system by an external effector, top-down elasticity analyses provide all the data needed for
a top-down control analysis of the system (11, 18). A top-down control
analysis was completed using the data from the present elasticity
analyses around 
m in old and
young hepatocytes. The results for cells in the resting state and in
State 4 are shown in Tables 1-3. The results were calculated using
mitochondrial respiration rates; similar elasticities and control
coefficients were obtained when calculations were based on total
cellular respiration rates. The elasticities to

m of the substrate
oxidation, phosphorylating, and proton leak subsystems are given in
Table 1. All of the data needed for the
calculation of elasticity and control coefficients can be obtained from

m and oxygen consumption
values in Fig. 2, A,
B, and
C. Values of each, and the inverse
slopes of the respective elasticity lines, are then used in the
published series of equations (18) for the calculation of control
coefficients.
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Table 1.
Overall elasticities to  m
for substrate oxidation, phosphorylating, and proton leak
subsystems in hepatocytes from old and young control mice
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The flux control coefficients of the three subsystems over the rate of
each of the subsystems are shown in Table
2,
A-C. The flux control coefficients describing the distribution of control over mitochondrial oxygen consumption
(JS) are shown
in Table 2A. The results from the
young control hepatocytes in the resting state indicate that the
substrate oxidation reactions (0.51; i.e., 51% of the control) and
phosphorylation reactions (0.45) exert most of the control over
JS, whereas the
remainder of the control is through the proton leak (0.04). These
results are similar to those obtained with hepatocytes from euthyroid
rats (24, 25); however, the amount of control exerted by the proton
leak is smaller here than with the latter. This is most likely due to
the respiration state in these cells under our resting incubation
conditions; it is possible that these hepatocytes are respiring at a
state closer to State 3. As mitochondrial respiration approaches State 3, the amount of control exerted by the leak over oxygen consumption approaches zero (11). Moreover, these metabolic control data are the
first from cells of mice, and these differences may indeed reflect a
species effect. Results from old mice, compared with those from young
controls, show an interesting shift in control over
JS away from
substrate oxidation reactions toward the phosphorylation and leak
reactions. In old cells, 54 and 12% of the control over resting
mitochondrial oxygen consumption are mediated through phosphorylation
and leak reactions, respectively. The corresponding values in young
control cells are 45 and 4%.
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Table 2.
Flux control coefficients over subsystem fluxes and concentration
control coefficients over  m
in hepatocytes from old and young control mice
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Results shown in Table 2B similarly
show that, in old cells compared with young, there is a shift in
control over the phosphorylation reactions away from substrate
oxidation reactions toward phosphorylation reactions and leak
reactions. Control by the substrate oxidation reactions and
phosphorylation reactions over the flux through the mitochondrial
proton leak reactions (Table 2C) in
old cells compared with young shows that there are decreases to roughly equal but opposite extents for these two blocks of reactions. Control
over leak flux by the leak reactions themselves remains high in both
old and young.
The concentration control coefficients of the three subsystems over the
intermediate in the system,

m, are shown in Table 2D. Control coefficients describe the
distribution of control by blocks of reactions in a system over the
amount of the intermediate in the system, and, unlike flux control
coefficients that sum to unity, concentration control coefficients sum
to zero. The values are also similar to those determined for
hypothyroid cells (25) and for euthyroid cells (11, 25). The results
for old and young cells are roughly similar and show that most of the control over the amount of the intermediate,

m, is exerted by the
substrate oxidation subsystem, and that the remainder of the control is
through the activity of the

m consumers: the proton leak
and phosphorylating subsystems.
As described by Brand et al. (6), it is possible to estimate the actual
number of ATP molecules synthesized by mitochondria for each oxygen
atom consumed (effective P/O ratio) and the distribution of control
(i.e., control coefficients) over the effective P/O by use of data such
as those described above. Because phosphorylation flux is measured as
the oxygen consumed to drive the phosphorylation reactions, the ratio
of JP divided by
JS provides the
fraction of oxygen consumption that is used to support phosphorylation, irrespective of the true value of the maximum P/O. The remaining fraction of the oxygen consumption
(JL/JS)
is used to support mitochondrial proton leak reactions. Thus the
effective P/O at any rate between State 3 and State 4 is this ratio
multiplied by the maximum P/O,
P/Omax. The effective P/O values,
based on mitochondrial oxygen consumption data, are shown in Table
3. In theory, the oxidation of glucose by
cells produces a maximum of 31 molecules of ATP per molecule of glucose
(27); this corresponds to a P/Omax
of 2.58. The values shown in Table 3 for mitochondrial respiration are
higher than the previously published values (6) and again support the
postulate that cells are metabolically positioned close to State 3 respiration.
Brand et al. (6) reported the derivation of flux control coefficients,
which quantitatively describe the control by the three blocks of
reactions over the effective P/O
These
equations were used with the flux control coefficients shown in Table 2
by use of values from the resting respiration states; the resulting
coefficients are shown in Table 3. Because these control coefficients
describe control over the fraction of the oxygen flux that is used to
drive ATP synthesis (i.e., over the ratio
JP/JS),
their values are independent of assumed or calculated values of
P/Omax.
Similar to the results for hepatocytes described earlier (6), the block
of reactions with the least amount of control is that of substrate
oxidation reactions, showing that increases in substrate supply on
their own produce only very small changes in the effective P/O. The
control coefficients for the leak reactions over the effective P/O are
relatively large and negative, indicating that increases in
mitochondrial proton leak reactions would cause substantial decreases
in the effective P/O. The coefficients from cells of old compared with
those of young mice differ in that the amounts of control by
phosphorylation and leak reactions are more than double in each case.
Control by substrate oxidation reactions remains low in old and young
cells. These results indicate that, in hepatocytes from old mice, the
efficiency of oxidative phosphorylation is more sensitive to changes in
the amount of mitochondrial proton leak and in the rate of ATP
synthesis and turnover reactions.
 |
DISCUSSION |
Our initial hypothesis was that increases in the mitochondrial proton
leak occur with aging as an effect of oxidative damage to the lipids of
the MIM. This damage then affects the integrity of the lipid bilayer
through altered lipid-lipid or lipid-protein interactions, the outcome
being an increased proton leak. Changes in the mitochondrial proton
leak with aging were expected to be rather small, but it was
anticipated that a kinetic approach, such as elasticity analysis, in
which the activities of blocks of reactions are assessed over a range
of metabolic "challenges," would identify and quantify any such
changes. Our findings do indeed show that the overall kinetics of the
mitochondrial proton leak are altered by age such that over a range of

m values, there is an
increased rate of oxygen consumed to balance the rate of the proton
leak. Chronic increases in mitochondrial proton leak may have negative
implications, because even minor increases are likely to initiate
compensatory increases in the activity of the proton pumps of the
electron transport chain to maintain protonmotive force. This may, in
turn, result in the increased production of free radicals at the level
of the electron transport chain. As discussed in more detail below, our
results do not indicate any changes in the kinetics of the substrate
oxidation reactions. The results do, however, show a marked decrease in
the ATP turnover reactions.
Changes in the overall kinetics of the mitochondrial proton leak are
consistent with the recent report of Hagen et al. (21), who studied
mitochondrial membrane potentials, cellular production of oxidants, and
levels of age-associated mtDNA deletions in rat hepatocytes.

m was assessed using
rhodamine 123 accumulation. Three distinct hepatocyte populations from
old rats were collected by centrifugal elutriation and were found to
have differing 
m values. One
population of cells was collected from the young rats. In the largest
population of old cells (~66% of cells in the preparation), membrane
potential was ~40% lower than that of the young cells. A smaller
subset of hepatocytes from the livers of old rats (~25% of the
cells) had only slightly lower fluorescence than those from young rats.
In the third subset, membrane potential was equal or greater than that
of the young cells. Their rhodamine 123 fluorescence results were
corroborated by quantitative determinations of potentials by assessing
mitochondrial uptake of radiolabeled tetraphenylphosphonium ion.
Membrane potential in young hepatocytes was 154.3 ± 20.4 mV; this
value is similar to that reported herein, 147 ± 3.4 mV. In their
three populations of old hepatocytes, values were 70.4 mV (66% of
cells), 92.6 mV (25% of cells), and 154.4 mV (remainder of cells). The
resting 
m in our old cells
was 149 ± 4.4 mV, which is clearly not different from our mean value
in young cells. Hagen et al. also examined resting oxygen consumption
rates of hepatocytes. Although their absolute values appear high, their finding that oxygen consumption was 36% lower in the largest fraction of hepatocytes compared with young cells supports our findings. Finally, in support of the oxidative stress theory, they found that
both the largest and the smallest fractions of cells produced significantly more oxidants than cells from young rats.
The very recent findings of Brookes et al. (10) also support our
findings. In their study on the effects of peroxynitrite on brain
mitochondria, they found that three sequential additions of 200 µM
peroxynitrite (initial concentration) to rat brain mitochondria (0.2 mg
protein/ml) significantly stimulated mitochondrial proton leak.
Cyclosporin A did not affect the stimulation, suggesting no involvement
of the mitochondrial permeability transition pore. However, the
stimulation was prevented by the vitamin E analog Trolox, supporting
the involvement of lipid peroxidation, a proposed mechanism of
peroxynitrite cytotoxicity. The authors link lipid peroxidation-mediated increases in mitochondrial proton leak to certain
neurodegenerative disorders thought to proceed via mechanisms of
mitochondrial oxidative damage.
The mechanism of proton leakage warrants discussion, particularly in
light of recent developments in this area. Potential mechanisms
underlying the mitochondrial proton leak are depicted in Fig.
4. Within the past year, two uncoupling
proteins (UCP2 and UCP3) were identified and found to be distinct from
the brown adipose tissue-specific uncoupling protein (UCP1) (4, 15, 44). These proteins explain at least some of the proton leak that has
been assessed in mitochondria from a variety of tissues. Thus far, none
of the three currently known uncoupling proteins have been found in
parenchymal cells, which constitute 95-97% of the cells isolated
using the techniques employed (2). Fleury et al. (15) report low levels
of UCP2 mRNA in liver; however, recent findings localize UCP2 to the
Kuppfer cells of the liver (31). Nevertheless, it is possible, and
likely, that there is an as-yet-unidentified uncoupling protein in the
parenchymal cells. Furthermore, it is possible that there are changes
in the amount and/or activity of such an uncoupling protein in
these cells. However, again, our underlying hypothesis, founded on a
substantial supportive literature, implicates free radical damage to
lipids, thus affecting the integrity of the bilayer at the lipid-lipid or lipid-protein interfaces.

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|
Fig. 4.
Proposed mechanisms for mitochondrial proton leak. Activity of redox
proton pumps of electron transport chain creates protonmotive force.
The latter drives ATP synthase activity. Mitochondrial proton leak,
regardless of its mechanism, allows protons to bypass ATP synthase, and
protonmotive force is dissipated. Thus additional oxygen is used by the
chain to maintain protonmotive force. The mechanism of the leak in
parenchymal cells of the liver is as yet unknown but may involve an
as-yet-unidentified uncoupling protein and/or bilayer lipid
composition of the mitochondrial inner membrane.
|
|
On the basis of previous reports of changes in the amounts and
activities of a wide range of enzymes involved in substrate oxidation
and specifically in components of the electron transport chain (3, 22,
43), we expected to observe changes in the kinetics of the substrate
oxidation reactions. We hypothesized that we might observe results
reflecting a decreased activity of this block of reactions.
Alternatively, it was reasoned that, as a result of the increased
proton leak, there may be a compensatory increase in the rate of the
substrate oxidation reactions in an effort to restore protonmotive
force to normal values; this would then accelerate the production of
reactive oxygen metabolites, which in turn would induce further
oxidative damage to cellular components. Our data (Fig.
2B) show clearly that any such
changes as measured under our conditions are quantitatively
insignificant. This does not indicate that free radical damage has not
occurred but shows only that this group of reactions responds normally to imposed changes in the rate of its activity and may indicate also
that the functioning of this block of reactions is at some level
protected.
Our results describing decreases in the proportion of resting oxygen
consumption of hepatocytes and altered kinetics of the phosphorylation
reactions were not anticipated. Beyond the extensive analyses of
respiratory control ratios in the literature (see for example Ref. 22),
data on age-related alterations in ATP synthesis and turnover reactions
are lacking. Thus it is difficult to speculate about the specific
mechanisms responsible for the decreased amount of oxygen used by cells
to support these reactions. As described in Fig. 1, ATP synthesis and
turnover mechanisms include ATP synthetic reactions, such as the
adenine nucleotide carrier and the phosphate transporter. This block of
reactions also includes all cellular ATP-consuming processes, such as
those involved in maintaining ion gradients across membranes (e.g., Ca2+-ATPase,
Na+-K+-ATPase)
and in protein, DNA, and RNA synthetic reactions. Consistent with the
oxidative stress theory, these findings may be related to known
age-related increases in oxidative damage to mitochondrial proteins and
DNA and should be examined further.
Importantly, the findings herein provide the first metabolic control
analysis of oxidative phosphorylation in relation to the metabolic
effects of aging. The data provide quantitative information about the
control over resting oxygen consumption, over other blocks of
reactions, and over 
m. In
old compared with young cells, there is a shift in control over resting
oxygen consumption away from the substrate oxidation reactions toward phosphorylation and leak reactions. Thus oxygen consumption of old
cells is more sensitive to changes in the rate of ATP turnover and in
mitochondrial proton leak rate. Similarly, there is a shift in control
over phosphorylation reactions away from substrate oxidation reactions
toward phosphorylation reactions and the leak in old cells.
In addition, the control coefficients describing the control over the
effective P/O denote that a greater amount of control is possessed by
the ATP turnover reactions and the leak in old hepatocytes compared
with young. This suggests an augmented capability of the latter blocks
of reactions to affect changes in the efficiency of oxidative
phosphorylation.
Overall, our findings, gathered from a relatively novel experimental
perspective, extend our understanding of the effects of aging on
oxidative phosphorylation in hepatocytes. The findings confer
additional support for the oxidative stress theory of aging. They
provide new quantitative data on the altered kinetics of the
mitochondrial proton leak and of ATP turnover reactions and show shifts
in metabolic control with aging.
 |
ACKNOWLEDGEMENTS |
The authors thank Rod Nicholls for assistance with the electron
microscopy of cells.
 |
FOOTNOTES |
This study was supported by grants from the Natural Sciences and
Engineering Research Council (NSERC) of Canada (M. E. Harper) and the
National Institutes of Health (RR-00167, R. Weindruch and J. J. Ramsey,
and PO1 AG-11915, R. Weindruch).
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: M. E. Harper, Dept. of Biochemistry,
Microbiology and Immunology, Faculty of Medicine, Univ. of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada K1H 8M5.
Received 15 January 1998; accepted in final form 23 April 1998.
 |
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