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From the
Received for publication, May 23, 2001, and in revised form, August 13, 2001
A mev-1(kn1) mutant of the nematode
Caenorhabditis elegans is defective in the cytochrome
b large subunit (Cyt-1/ceSDHC) in complex II of the
mitochondrial electron transport chain. We have previously shown that a
mutation in mev-1 causes shortened life span and rapid
accumulation of aging markers such as fluorescent materials and protein
carbonyls in an oxygen-dependent fashion. However, it
remains unclear as to whether this hypersensitivity is caused by direct
toxicity of the exogenous oxygen or by the damage of endogenous
reactive oxygen species derived from mitochondria. Here we report
important biochemical changes in mev-1 animals that serve
to explain their abnormalities under normoxic conditions: (i) an
overproduction of superoxide anion from mitochondria; and (ii) a
reciprocal reduction in glutathione content even under atmospheric
oxygen. In addition, unlike wild type, the levels of superoxide anion
production from mev-1 mitochondria were significantly elevated under hyperoxia. Under normal circumstances, it is well known
that superoxide anion is produced at complexes I and III in the
electron transport system. Our data suggest that the
mev-1(kn1) mutation increases superoxide anion production
at complex II itself rather than at complexes I and III. The
mev-1 mutant also had a lactate level 2-fold higher than
wild type, indicative of lactic acidosis, a hallmark of human
mitochondrial diseases. These data indicate that Cyt-1/ceSDHC plays an
important role not only in energy metabolism but also in superoxide
anion production that is critically involved in sensitivity to
atmospheric oxygen.
Mitochondria are specialized organelles whose primary function is
to synthesize ATP via oxidative phosphorylation in aerobic eukaryotes
(1). During this process, electrons are transferred ultimately to
oxygen after their passage through four membrane-bound complexes.
Electrons enter the electron transport system through either complex I
(NADH-CoQ1 oxidoreductase) or
complex II (succinate-CoQ oxidoreductase). Via two, single-electron
reductions, they are transferred to CoQ (coenzyme Q or ubiquinone),
thereby reducing CoQ first to ubisemiquinone and then to ubiquinol
(dihydroubiquinone). At complex III (CoQH2-cytochrome c oxidoreductase) molecules of ubiquinol undergo two
sequential and spatially separate one-electron oxidations, a process
called Q cycle. These reducing equivalents are then transferred to the remainder of the electron transport chain: cytochrome c,
complex IV (cytochrome c oxidase), and finally to oxygen.
Mitochondrial deficiencies cause a variety of diseases, including human
congenital neurodegenerative diseases like MELAS (mitochondrial
encephalomyopathy, lactic acidosis, and stroke-like episodes), MERRF
(myoclonic epilepsy with ragged red fibers), KSS (Kearns-Sayre
syndrome), CPEO (chronic progressive external ophthalmoplegia), NARP
(neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally
inherited Leigh syndrome), and LHON (Leber hereditary optic neuropathy)
(2-4). Many of the ultimate manifestations of these diseases are
triggered by a metabolic imbalance known as lactic acidosis, which is
characterized by a high lactate/pyruvate ratio and is a characteristic
feature of a variety of other metabolic disorders in addition to
mitochondrial diseases. In contrast to the well defined pathologies
resulting from complex I, III, and IV deficiencies, the effects of
complex II defects in humans are not as completely understood (5-13). A complex II deficiency has been described that is associated with a
mitochondrial disease called Leigh syndrome and resulted in metabolic
changes leading to lactic acidosis (5, 6). Another mutation in the
flavoprotein subunit of complex II has recently been shown to cause
late onset optic atrophy, ataxia, and myopathy (10, 11). Finally,
individuals with an inherited propensity for vascularized head and neck
tumors (i.e. paragangliomas) have been recently demonstrated
to contain one of several mutations in the small and large subunits of
cytochrome b in succinate-ubiquinone oxidoreductase (14,
15). In addition to its role in electron transport, the succinate
dehydrogenase (SDH) moiety of complex II plays an essential role in the
trichloroacetic acid cycle, catalyzing the conversion of succinate to
fumarate. Complex II is composed of four subunits, named SDHA through
SDHD in Escherichia coli or human and SDH1 through SDH4 in
yeast. SDHA/SDH1 is a flavoprotein subunit, and SDHB/SDH2 is an
iron-sulfur protein subunit. Two other hydrophobic membrane-anchored
subunits (consisting of large and small subunits of cytochrome
b) are named SDHC/SDH3/CybL and SDHD/SDH4/CybS. We will
refer to the Caenorhabditis elegans orthologs as
Cyt-1/ceSDHC through ceSDHD.
The electron transport system is also the major endogenous source of
reactive oxygen species (ROS) such as superoxide anion (O We have previously described a mev-1(kn1) mutant of the
nematode C. elegans that has a missense mutation in
Cyt-1/ceSDHC in mitochondrial complex II (19). The mutant, isolated by
screening for hypersensitivity to oxygen, ages precociously (19, 20). We have also reported that the mev-1 mutant accumulates
markers of aging (e.g. fluorescent materials and protein
carbonyls) more rapidly than wild type (21-23) at high oxygen
concentrations. In addition, the mev-1 mutant has much lower
succinate-cytochrome c oxidoreductase activity than wild
type (19). However, the reason the mev-1 mutant is
hypersensitive to hyperoxia as well as the role of Cyt-1/ceSDHC was
completely unclear. We now demonstrate that mev-1 animals
manifest a series of biochemical defects that serve to explain their
oxygen-hypersensitive cellular and organismal mutant phenotypes. In
addition, the data also suggest that, contrary to the current sentiment
in the scientific literature, significant amounts of superoxide anion
may be produced at complex II.
Mitochondrial Isolation--
To prepare stage-synchronized
animals of either wild type (N2: Bristol strain) or
mev-1(kn1), isolated embryos were cultured on nematode
growth medium (NGM) at 20 °C under atmospheric (21%) or
hyperoxic (40%) conditions. Young adult animals were harvested 72 h later by centrifugation and were homogenized (10% w/v) in isolation buffer (210 mM mannitol, 70 mM
sucrose, 0.1 mM EDTA, and 5 mM Tris-HCl, pH
7.4) with a Teflon homogenizer. Mitochondria were isolated by
differential centrifugation (24) and suspended in Tris-EDTA buffer (0.1 mM EDTA, 50 mM Tris-HCl, pH 7.4).
Submitochondrial particles (SMP) were obtained by sonicating
freeze-thawed mitochondria twice for 20 s with 1-min intervals in
a model U200S sonicator (IKA Labortechnik). SMP were washed
twice and suspended in isolation buffer.
Biochemical Analyses--
The activities of complex I plus III
(NADH-cytochrome c oxidoreductase) and complex II plus III
(succinate-cytochrome c oxidoreductase) in mitochondria
isolated from wild-type and mev-1 young adult animals were
measured as described (24).
Mitochondrial Superoxide Anion Generation Assay--
Superoxide
anion generation was measured using the chemiluminescent probe MCLA
(cypridina luciferin analog,
2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2- GSH Assay--
The levels of total thiol groups (total SH) and
reduced glutathione (GSH) in whole lysates from wild-type and
mev-1 young adult animals were measured by the rate of
formation of 5,5'-dithio-(2-nitrobenzoic acid) as described (25).
Metabolite Analyses--
Young adult animals were cultured under
atmospheric (21%) or hyperoxic (50%) conditions for 16 h,
and whole lysates were prepared as described below. Animal
lysates were collected by centrifugation at 10,000 × g for 10 min. The pellets were suspended in an equal volume
of 10% trichloroacetic acid and homogenized with ultra-Turrax T8 (IKA
Labortechnik) on ice and then sonicated with a Sonifier 450 (Branson).
The homogenates were clarified by centrifugation at 10,000 × g for 10 min. The supernatants then were neutralized with 4 N KOH and centrifuged at 10,000 × g for 10 min. Lactate and pyruvate were measured using Sigma diagnostic kits.
Citrate, succinate, and glutamate were measured with an enzymatic
bioanalysis/food analysis kit (Roche Diagnostics). ATP was measured
using an ATP bioluminescence assay kit CLS II (Roche Diagnostics).
Confirmation of a Complex II Defect in mev-1 Mutants--
We first
measured the activities of complex I and complex II in wild-type and
mev-1 genetic backgrounds to directly confirm the genetic
evidence that the mev-1 mutant is normal for NADH-cytochrome c oxidoreductase (complex I plus III) activity but has a
severely reduced succinate-cytochrome c oxidoreductase
(complex II plus III) activity (Table I).
The activity of succinate-CoQ oxidoreductase (complex II) in
mev-1 mutant mitochondria was also markedly lower than that
in wild type (data not shown). By extension, these data suggest that
the various biochemical manifestations of the mev-1 mutation
derive from a defect in the electron transfer between complex II and
complex III. Because Northern and Western blots revealed roughly equal
mRNA and protein levels of Cyt-1/ceSDHC in wild-type and mutant
animals (data not shown), the mev-1 mutation most likely
compromises enzyme activity per se as opposed to affecting complex assembly.
Production of Superoxide Anion from Mitochondria Is Elevated in
mev-1 Mutants--
The specific mechanism by which a complex II
deficiency causes hypersensitivity to exogenous oxygen in
mev-1 mutants has not been directly addressed. There is no
evidence in the literature to suggest that complex II contributes
directly to ROS production. Indeed, it is currently thought that the
majority of ROS are produced in mitochondria at complex III (16-18).
To determine the effects of the mev-1 mutation on
mitochondrial ROS production, we examined mitochondria from
mev-1 and wild-type animals cultured under atmospheric and
hyperoxic conditions. The levels of superoxide anion production were
found to be approximately 2-fold higher in intact mitochondria and SMP
from mev-1 animals as compared with wild type under
atmospheric oxygen (Fig. 1). SMP
were employed because they are known to be depleted in the
mitochondrial superoxide dismutase (MnSOD) that transforms superoxide
anion to hydrogen peroxide.
As a next step, we examined the effect of hyperoxia on superoxide anion
production from intact mitochondria (Fig.
2). The level of superoxide anion
production from intact mev-1 mitochondria (without
stimulation by exogenous succinate and antimycin A) was higher than in
wild type and was significantly increased under hyperoxia (40%
O2). Specifically, it was ~3.4-fold higher than the wild
type under 40% O2 and ~1.6-fold higher than
mev-1 under 21% O2. On the other hand,
superoxide anion production from the wild-type mitochondria was
relatively independent of oxygen concentration. These results suggest
that the mutation in this complex II-encoding gene causes cell damage
and precocious aging by the endogenous generation of ROS in
mitochondria rather than by the direct toxicity of exogenous
oxygen.
We further examined superoxide anion production as it is influenced by
succinate, a complex II substrate, and antimycin A, a Q cycle (complex
III) inhibitor (Fig. 2). Both succinate and antimycin A stimulated
superoxide anion production. The proportional increase of the succinate
stimulation was approximately the same for wild-type and
mev-1 mutants, although the absolute levels were
significantly higher in mev-1 mutants. In addition,
inclusion of antimycin A to succinate-stimulated mitochondria increased superoxide anion production much more in wild-type than in
mev-1 mutants, particularly under hyperoxia.
Glutathione Concentrations Are Reduced in mev-1 Mutants--
We
also measured total cellular levels of GSH. GSH can either act directly
as an antioxidant or as a substrate for the ROS detoxifying enzyme
glutathione peroxidase making it an important defense against ROS (26,
27). Only 10-15% of the total cellular GSH is normally found inside
the mitochondrial matrix (26). An age-associated increase in superoxide
anion and hydrogen peroxide generation by mitochondria and an
age-related decline in GSH content are observed in senescent organisms
including both mammals and insects (27, 28). Elevated mitochondrial ROS
could act as a sink to eventually reduce or deplete total cellular GSH
levels, ultimately rendering the entire cell more susceptible to the
effects of mitochondrially generated ROS. This in turn could lead to
the dual phenotypes of oxygen-hypersensitive precocious aging that are
the hallmark phenotypes of mev-1 mutants. Indeed, total
cellular GSH levels were decreased ~30% in mev-1 mutants
when compared with wild type (Fig. 3).
Because a thiol group constitutes the reactive moiety of GSH, we also
confirmed that total thiol content in mev-1 mutants was
experimentally identical to wild-type levels (Fig. 3). Thus, it is
likely that the Cyt-1/ceSDHC deficiency leads to an overproduction of
mitochondrial ROS, which in turn reduces GSH levels in both cytosol and
mitochondria. In concert these biochemical abnormalities render
mev-1 animals hypersensitive to hyperoxia. Alternatively,
the reduced GSH levels in mev-1 extracts may have resulted
from some unknown metabolic changes in mev-1 mutants
versus wild type. A stoichiometric comparison of superoxide anion and GSH concentrations would distinguish these alternatives. Such
an analysis was not attempted because GSH and the superoxide anion were assayed using different protocols that employed whole lysates in one case and isolated mitochondria in another. However, a
variety of data suggest that GSH levels vary in direct response to
superoxide anion scavenging rather than to metabolic suppression (26).
Cyt-1/ceSDHC Affects the Levels of Trichloroacetic Acid Cycle
Intermediates--
It is possible that the complex II deficiency by
the Cyt-1/ceSDHC mutation in mev-1 mutants might impede flow
through the trichloroacetic acid cycle and alter the lactate/pyruvate
ratio, resulting in lactic acidosis. Indeed, although pyruvate
concentrations were not significantly different in wild type and
mev-1 mutants, there was over twice as much lactate in
mev-1 animals, i.e. the lactate/pyruvate ratio in
mev-1 mutants was ~1.6-fold higher than that in wild type
(Fig. 4A and Table
II). Both citrate and succinate levels
were normal in mev-1 animals; however, glutamate was present in reduced amounts in mev-1 animals at statistically
significant levels (Fig. 4B). The decrease in glutamate
suggests that the trichloroacetic acid cycle intermediate
ATP Levels Are Normal in mev-1 Mutants--
Given the complex II
deficiency in mev-1 animals and its various biochemical
consequences, it was surprising that ATP levels were experimentally
identical in wild type and mev-1 mutants under atmospheric
oxygen (Fig. 4C). Even under 50% oxygen the slight reduction in ATP was not statistically significant (Fig.
4C). It seems likely that mev-1 animals rely more
heavily on glycolysis for energy acquisition, thus explaining the
elevated lactate levels (Fig. 4A). However, it is also
possible that ATP consumption is decreased in mev-1 because
of some sort of global decrease in the metabolic rate that acts to
counterbalance the compromised ATP generation in mev-1 animals.
Mitochondria are widely accepted as the major intracellular site
of ROS production because of the inappropriate single-electron reduction of diatomic oxygen to superoxide anion (16, 18). Because once
generated ROS react indiscriminately with a wide variety of cellular
constituents, they figure prominently in theories seeking to explain
the etiology of aging and neurodegenerative diseases (16-18, 29). As
such, it is important to understand the precise mitochondrial location
and mechanism of superoxide anion generation. Work with the
mev-1(kn1) mutation of the nematode C. elegans
may prove useful in this regard. As articulated in the Introduction,
mev-1 mutants are hypersensitive to oxidative stress and age
precociously (19-23). This has been attributed to a missense mutation
in the gene encoding Cyt-1/ceSDH3, a large subunit of cytochrome
b, that compromises complex II activity (19).
We now demonstrate that superoxide anion production is significantly
higher in mev-1 animals, particularly under hyperoxia (Figs.
1 and 2). Along with the reduced GSH levels we have observed in
mev-1 animals (Fig. 3), these data provide the biochemical connection that links the molecular defect in complex II to the organismal phenotypes of ROS hypersensitivity and precocious aging. Perhaps more importantly, the data bear on the question of precisely where in the mitochondria and how superoxide anion is generated.
It is generally believed that most superoxide anions are produced at
complex III (16-18). Specifically, the free radical ubisemiquinone is
generated during the Q cycle, a series of events that includes a
two-step oxidation from ubiquinol to ubisemiquinone to ubiquinone. Ubisemiquinone is alternatively capable of transferring electrons to
diatomic oxygen, forming superoxide anion. Given this, how can the
increased superoxide production in mev-1 animals be
explained? First, the increase may be an indirect effect of the
mev-1 mutation that uncouples some aspect of electron
transport and results in increased superoxide anion at complex III
itself. Second, the increase may be a direct effect of the
mev-1 mutation such that superoxide anion is produced at
complex II. Two variations on this second scenario could be envisioned.
The first is that the complex II defect could affect electron flow such
that there is a probable increase of electrons leaking directly from
complex II to molecular oxygen. The second is that the mutation results in increased superoxide anion generation because of semiquinone involvement; specifically, the mutation could result in a premature release of the free radical semiquinone before its reduction to ubiquinol.
Several lines of evidence suggest that the mev-1 mutation
results in superoxide anion production at complex II itself rather than
exerting an indirect effect on complex III. Interpretation of inhibitor
data is fraught with danger; however, the antimycin A results (Fig. 2)
lend support to the notion that superoxide anion can be produced at
complex II, particularly in mev-1 animals. Specifically,
disruption of the complex III-associated Q cycle by antimycin A had
comparatively little effect on superoxide anion production in
mitochondria derived from mev-1 animals, suggesting that
production occurred at some location other than at complex III.
Conversely, the large antimycin A-mediated increase in wild type is in
keeping with other evidence implicating complex III as the source of
most superoxide anions under normal conditions. That succinate
stimulated superoxide anion production more in mev-1 mutants
than wild type is also consistent with our contention that the
mev-1 mutation results in high levels of superoxide anion production at complex II. Similarly, the relative inability of antimycin A to exacerbate the increases mediated by succinate in
mev-1 (but not wild type) also suggests that complex II is the source of most superoxide anion in mev-1 mutants.
In addition to the inhibitor data, the specific nature of the
mev-1 mutation is consistent with a direct, complex
II-mediated production of superoxide anion. Specifically, site-directed
mutagenesis has been employed in E. coli to demonstrate that
amino acid residues 17-33 are likely the ubiquinone-binding sites in
the E. coli SDHC to which Cyt-1/ceSDHC is the probable
ortholog (30). This corresponds to amino acids 59-76 in Cyt-1/ceSDHC.
We have shown previously that the mev-1 mutant has a
missense mutation that substitutes glutamic acid for glycine at residue
71 (19). Therefore, it seems reasonable to speculate that this mutation
may result in premature release in semiquinone, thereby enabling
superoxide anion formation via its subsequent interaction with
molecular oxygen. Whatever the case, it seems likely that the
mev-1 mutation exerts an allele-specific effect, that is the
missense mutation at position 71 may affect the quinone-binding site
whereas other, more severe mutations would not elevate superoxide anion production.
It also seems reasonable to posit that some superoxide anion
production might occur at complex II even in wild-type
mitochondria. In particular, the observation that succinate, the
substrate for complex II, increased superoxide anion production in
wild-type mitochondria suggests that complex II might even be a
secondary source of superoxide anion production in wild type.
Many reports have implicated that mitochondrial function is
reduced in the aging process (1, 16-18, 29). Several studies have
demonstrated age-linked declines in the activities of mitochondrial electron transport enzymes NADH-cytochrome c oxidoreductase
(complex I plus III), succinate-cytochrome c oxidoreductase
(complex II plus III), ubiquinol-cytochrome c oxidoreductase
(complex III), cytochrome c oxidase (complex IV), and ATP
synthase (complex V) in human skeletal muscle (31). Mitochondria are
the major source of ROS and are also the first compartment in the cell
that is damaged by these ROS. Indeed, aging is accompanied by a
decrease in mitochondrial function. That has led to the hypothesis that a deficit in energy metabolism and an increase in ROS production could
be a cause of aging. This hypothesis is strongly supported by the
model of short-lived mev-1 mutants.
In summary, we have shown that mev-1 animals manifest two
distinct biochemical pathophysiologies, namely superoxide anion overproduction from mitochondrial complex II itself (with an attendant decrease in glutathione levels) and metabolic changes with lactic acidosis by the mutation in Cyt-1/ceSDHC subunit (Fig.
5). The metabolic changes in
mev-1 mutants are biochemical and metabolic features
associated with human mitochondrial diseases. The superoxide anion
overproduction ultimately results in (or strongly contributes to) the
exogenous oxygen-dependent mev-1 phenomena:
shortened life span and damage to lipids and proteins (rapid
accumulation of fluorescent materials and protein carbonyls) that might
be correlated with the pathogenesis of mitochondrial diseases and precocious aging. Elevated ROS by mitochondrial dysfunction is also
thought to lead to neurodegeneration, cell death, and damage to nuclear
and mitochondrial DNA. The mechanisms are currently poorly understood.
Therefore, studies of those cellular and organismal sequelae in
mev-1 mutants might elucidate the pathogenic mechanisms of
mitochondrial disease and aging.
We thank K. Kita for critical comments on the manuscript.
*
This work was supported, in part, by a grant-in-aid for
aging research from the Ministry of Health and Welfare, by a
grant-in-aid for scientific research from the Ministry of Education,
Science, Sports and Culture, Japan, and by a grant from Tokai
University Research Project.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. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, August 29, 2001, DOI 10.1074/jbc.M104718200
The abbreviations used are:
CoQ, coenzyme Q or ubiquinone;
SDH, succinate dehydrogenase;
ROS, reactive
oxygen species;
SMP, submitochondrial particles;
MCLA, cypridina luciferin analog,
2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-
A Defect in the Cytochrome b Large Subunit in Complex
II Causes Both Superoxide Anion Overproduction and Abnormal Energy
Metabolism in Caenorhabditis elegans*
,,,,,
Department of Molecular Life Science and
§ Department of Physiology, Tokai University School
of Medicine, Ishehara, Kanagawa 259-1193, Japan and ¶ Department
of Biology, Texas Christian University, Fort Worth, Texas 76129
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
]pyrazin-3-one). Forty micrograms of either intact mitochondria or SMP were added to 1 ml of assay buffer (50 mM HEPES, pH 7.4, 2 mM
EDTA) containing 0.5 mM MCLA. For the measurement of
superoxide anion generation from mitochondria with substrate and
inhibitor, 1.5 mM succinate as a complex II substrate was
added in the mitochondrial solution, and 100 µM antimycin
A as a complex III inhibitor was added after addition of the
mitochondria and succinate. The solution was placed into the photon
counter with a H-R550 photomultiplier (Hamamatsu Photogenic Co. Ltd.)
and measured at 37 °C. The rates of superoxide anion production were
expressed as counts per second. The amount of superoxide anion
production was calculated by subtracting the optical density of samples
in the presence of 10 µg/ml bovine Cu,Zn-superoxide dismutase from
that in the absence of Cu,Zn-superoxide dismutase.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Activities of NADH-cytochrome c oxidoreductase and succinate-cytochrome
c oxidoreductase in wild-type and mev-1 animals under atmospheric
oxygen
View larger version (23K):
[in a new window]
Fig. 1.
Superoxide anion production in mitochondria
or submitochondrial particles isolated from wild-type or
mev-1 young adult animals. Each value reported is
the mean ± S.D. of four different experiments. *,
p < 0.05; ***, p < 0.0005 by paired
Student's t test. Mito, mitochondria.
View larger version (31K):
[in a new window]
Fig. 2.
The effect of hyperoxia on superoxide anion
production. Wild-type or mev-1 young adult animals were
cultured from embryo stage for 72 h under atmospheric oxygen (21%
O2) or hyperoxia (40% O2). Superoxide anion
production from wild-type and mev-1 mitochondria was
measured in the presence or absence of 1.5 mM succinate
and/or 100 µM antimycin A. Each value reported is the
mean ± S.D. of three different experiments. For each value as
compared with wild type under 21% O2: **,
p < 0.005, and ***, p < 0.0005. For
mev-1 mutants under 40% O2 as compared with
mev-1 mutants under 21% O2: 
,
p < 0.05; , p < 0.005. Student's
t tests were done on all quantitative analyses.
View larger version (19K):
[in a new window]
Fig. 3.
The levels of total thiol groups and GSH in
whole lysates from wild-type or mev-1 young adult
animals. The values represent the mean ± SD. of four
different experiments. ***, p < 0.0005 by paired
Student's t test. Total SH, total
thiol groups.
-ketoglutarate, a precursor of glutamate, is reduced in its content.
On the other hand, in the patient with complex II and SDH defects,
urine levels of both succinate and -ketoglutarate were significantly
increased (5). An SDH defect may result in a block in the oxidation of
succinate to fumarate with accumulation of succinate. Given the
elevated lactate levels in mev-1 mutants, this finding
suggests that the flow of pyruvate into the trichloroacetic acid cycle
may be reduced. It is possible that these metabolic defects contribute
to the mev-1 phenotypes.
View larger version (19K):
[in a new window]
Fig. 4.
Changes in energy metabolism in
mev-1 mutants. Wild-type or mev-1
young adult animals were cultured under atmospheric oxygen (21%
O2) or hyperoxia (50% O2) for 16 h.
Concentrations of lactate and pyruvate (A), contents of
trichloroacetic acid cycle intermediates, citrate, succinate, and
glutamate (B), and ATP content (C) in whole
lysates of these animals were measured as described under
"Experimental Procedures." Each value reported is the mean ± S.D. of three different experiments. *, p < 0.05 and
**, p < 0.005 by paired Student's t
test.
The lactate to pyruvate ratio (L/P ratio) in wild-type and mev-1
mutants under either atmospheric oxygen (21%) or hyperoxia (50%)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (34K):
[in a new window]
Fig. 5.
Model of Cyt-1/ceSDHC subunit of complex II
deficiency in mitochondrial diseases and aging. The Cyt-1/ceSDHC
subunit deficiency causes mitochondrial superoxide anion (O
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed. Tel.:
81-463-93-1121 (Ext. 2650); Fax: 81-463-94-8884; E-mail:
nishii@is.icc.u-tokai.ac.jp.
ABBREVIATIONS
]pyrazin-3-one.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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