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Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan
1Correspondence: Tokyo Metropolitan Institute of Gerontology, 35–2 Sakaecho, Itabashiku, Tokyo, 173-0015 Japan. E-mail: hondas{at}center.tmig.or.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: life extension mutant • Mn-SOD • C. elegans • nematode
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, it has been postulated that a gene network exists
to determine the life span of a species (2)
. Life extension mutants of
the nematode Caenorhabditis elegans have recently been
isolated, and the gene network responsible for its longevity has been
unraveled (3)
, providing support for this hypothesis. Mutants of each
of two genes that are components of insulin-like signaling
transduction—daf-2, a homologue of the member of the
insulin receptor family (4)
and age-1, a homologue of the
mammalian phosphatidylinositol-3-OH kinase (5)
—display the life
extension (Age)2
phenotype (6
,7
) as well as the constitutive dauer formation (Daf-c)
phenotype (8
9
10)
. Animals proceed through four larval stages (L1-L4)
to become adults. However, when the food supply is limited and
population density is high, animals at the L1 stage proceed to the
dauer larva at the L2 stage. The dauer larva is a developmentally
arrested dispersal stage; it is resistant to adverse environmental
conditions and is adapted for long-term survival (8)
. It seems that the
dauer stage is non-aging, since the post-dauer life span is not
affected by a prolonged dauer stage of up to 2 months (11)
.
A number of genes that regulate dauer formation have been identified.
These genes have been assembled into regulatory pathways (8)
. Mutants
of some genes do not undergo dauer formation under conditions where the
food supply is limited and population density is high (Daf-d
phenotype), whereas mutants of other genes display the Daf-c phenotype
under normal conditions. Temperature-sensitive Daf-c mutants of the
daf-2 gene, which are located in the downstream positions of
the pathways, develop reproductively and display the Age phenotype at
permissive temperatures that do not induce dauer formation. In
contrast, Daf-c mutations in genes located in the upstream positions of
the pathways such as daf-1, daf-7, and
daf-11, including transforming growth factor-ß signaling,
do not affect the adult life span (6
, 12
). The simplest interpretation
of these observations is that the efficient life maintenance mechanism
of long dauer survival uncouples from other dauer formation mechanisms
and is inappropriately expressed in adult daf-2 mutants at
permissive temperatures, resulting in adult life span extension (6
,
12
).
The Daf-d mutation in the daf-16 gene, a homologue of an
HNF3/forkhead transcription factor (13
, 14
), and the Daf-d mutation in
the daf-18 gene, suppress the Age phenotype of
daf-2 mutants (6
, 12
). This indicates that insulin-like
signaling controls longevity by regulating the activity of DAF-16
transcription factor. Therefore, it seems likely that the insulin-like
signaling pathway does not change the activity of preexisting enzymes,
but instead transcriptionally activates the expression of target genes
that specify the efficient life maintenance mechanism.
It has been shown that the spe-26 mutant displays the Age
phenotype as well as reduced sperm formation (15)
. Another group of Age
mutants are those in a set of clk genes (clk-1, clk-2,
clk-3, and gro-1), which display an altered biological
timing phenotype (16)
. clk-1 is a homologue of yeast
CAT5/COQ7 (17)
, which is involved in gluconeogenesis (18)
and the
biosynthesis of ubiquinone, a substance that regulates energy
production in the mitochondria (19)
. The maximum period of time by
which life span is extended in a clk-1 mutant is only
~40%. However, the mean life span of the double mutant
daf-2;clk-1 is more than five times the life span of the
wild type (16)
. This indicates that the daf-2 insulin-like
signaling pathway and the clk-1 pathway interact in
determining the adult life span.
The aging theories that have been proposed thus far can be investigated
by examining the properties of these mutants to determine whether the
gene network in C. elegans supports or contradicts them. The
free radical theory of aging is attracting considerable attention (20
,
21
). Reactive oxygen species (ROS) such as
O2-.,
H2O2, and
OH., are generated during cellular metabolism,
especially during mitochondrial energy production (22)
. These ROS in
turn cause oxidative damage to DNA (23)
and proteins (24)
. This theory
of aging proposes that although the defense systems that enzymatically
detoxify these ROS (22)
and repair the oxidative damage (25)
have
evolved, not all ROS are detoxified; oxidative damage caused by the ROS
not caught by the defense systems accumulates to cause deleterious
consequences in senescence. The importance of oxidative stress in the
aging processes is evidenced by the observation that overexpression of
antioxidant defense enzymes in Drosophila extends life span
(26
, 27
).
We have tried to find the genetic pathway(s) that alleviates oxidative
stress in the longevity gene network of C. elegans. We have
screened the Age mutants associated with the oxidative stress
resistance phenotype, referred to as Oxr phenotype. The Oxr phenotype
may be determined by pathways involved in the detoxification of ROS.
These include superoxide dismutase (SOD), catalase, glutathione, and
systems that repair oxidative damage. SOD is a major enzyme that
protects against oxidative stress by catalyzing the removal of
O2-. (22)
, a central ROS
involved in the generation of various toxic ROS. In eukaryotes, there
are three types of SODs: cytosolic CuZn-SOD, extracellular CuZn-SOD,
and Mn-SOD, an enzyme located in the mitochondria, the major site of
O2-. generation (22)
. There are
several genes in C. elegans that encode SOD enzymes:
sod-1 encodes cytosolic CuZn-SOD (28)
whereas
sod-2 and sod-3 each encodes Mn-SOD (29
30
31)
.
Here we demonstrate the pattern of expression of sod genes
and its association with the Oxr phenotype among the Age mutants of
C. elegans.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The N2 Bristol strain was used as the wild
type. Synchronous populations of C. elegans in the egg, L1,
L2, L3, L4, and young adult stages were isolated at 20°C (33)
. The strains used in this study were:
Linkage group (LG) I: daf-16(m26);
LG II: fer-15(b26ts), age-1(hx542); fer-15(b26ts), age-1(hx546); fer-15(b26ts);
LG III: daf-7(e1372), daf-2(e1370ts), daf-2(sa189ts), daf-2(sa193ts), clk-1(e2519);
LG IV: daf-18(e1375), daf-1(m40ts), spe-26(hc138);
LG V: daf-11(m47).
Oxidative stress resistance assay
Approximately 20 hermaphrodites of C. elegans at the
L4 stage were placed in a solution that consisted of 50 mM paraquat
(PQ, Sigma, St. Louis, Mo.) or 20 mM menadione (Sigma), 0.2 ml S
medium, and OP-50 1 x 107/ml in 15 mm
diameter dishes (4-well Nunclon multiplates; Intermed, Roskilde,
Denmark). The animals were then exposed to 98% oxygen in an airtight
plastic chamber, as described previously (34)
, at 20°C. The chamber
was opened every 12 h, when the number of surviving animals were
counted and the gas (98% oxygen) was replaced. Animals that did not
move for 30 s after gentle mechanical touch were scored as dead.
Animals that had died by hatching of progeny inside the uterus were not
counted.
Preparation of dauer larva
Two Daf-c mutants, daf-2 (e1370) and daf-7
(e1372), were cultured at a nonpermissive temperature (25°C) for
2 wk, then treated with 1% sodium dodecyl sulfate for 30 min. The
dauer larva were washed with distilled water three times. The control
adult animals were obtained: both the daf-2 and
daf-7 mutants were cultured at a permissive temperature
(20°C) until the L4 stage, then cultured at 25°C for 1 day.
Northern blot analysis
Messenger RNA (mRNA) was isolated using the
guanidinium-acid-phenol-chloroform method (35)
and oligo(dT)-cellulose
spun columns (Amersham Pharmacia Biotech, Uppsala, Sweden) from
C. elegans at the young adult stage. Two micrograms of mRNA
was separated on 1.2% agarose-formaldehyde gels and blotted onto a
nylon membrane (Magna Graph, MSI, Mass.). The relative amount of mRNA
was judged by hybridization with a C. elegans ribosomal
protein, rp21c cDNA probe (EcoRI/SmaI
fragment of pPD33.24). The C. elegans sod-2 cDNA probe was
the KpnI/Sac-I fragment of BTK65–1 (29)
. The C.
elegans catalase cDNA probe was the EcoRI fragment of a
CeCAT (X82175) clone. The cDNA probes of C. elegans sod-1
and sod-3 cDNA were synthesized by reverse
transcription-polymerase chain reaction (RT-PCR) (Titan One Tube RT-PCR
System, Boehringer Mannheim, Mannheim, Germany) from total RNA of
C. elegans; the oligo DNA primers used for sod-1
were 5'-AGGTCGAAGCCGCTCAAAAAA-3' and 5'-ATTGTGTGGAGATTCAGAGA, and those
used for sod-3 were 5'-AATGCTGCAATCTACTGCTC-3' and
5'-AGCGTTTTAAACTACATCTG-3'. The cDNA was amplified (35 cycles;
94°C, 1 min; 55°C, 1 min; 72°C, 1 min) and the PCR products were
subcloned into the pGEM-T vector (Promega, Madison, Wis.) for
amplification. Hybridization was carried out in a solution that
consisted of 5 x SSPE, 10 x Denhardt's reagent, 50%
formamide, 1.4% sodium dodecyl sulfate, and 0.1 mg/ml herring sperm
DNA with 32P-labeled probes at 42°C (36)
. After
hybridization, the membrane was washed and the degree of hybridization
was estimated by using a Fujix BAS 2500 Laser Image Analyzer (Fuji
Film, Kanagawa, Japan).
RT-PCR analysis
Total RNA was isolated by the guanidinium-acid-phenol-chloroform
method (35)
. For RT-PCR, 1–100 pg RNA was used as the template. The
oligo DNA primers used in the Northern blot analysis of
sod-1 and sod-3 were used for the RT-PCR. The
oligo DNA primers used to detect PCR product were as follows:
sod-2 (5'-CTTCAAAACACCGTTCGCTG-3' and
5'-CAGTGGAACAAGTCCAGTT-3'); rp21c (5'-GCTTGCGTCTACCTGCTC-3'
and 5'-TCCGGAAGAGACAGAAGTGA-3'). The primer sets used for the
amplification of sod-2 did not amplify the cDNA of
sod-3 and the primer sets used for sod-3 did not
amplify the cDNA of sod-2. The PCR products were amplified
with the following amplification profile for 35 cycles: denaturation
for 1 min at 94°C, annealing for 1 min at 55°C, and extension for 1
min at 72°C.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, under hyperoxia (98% oxygen). Although hyperoxia itself did not
have a lethal effect on the wild type and mutants in this study over 4
days (data not shown), it did stimulate PQ toxicity (Table 1
).
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Table 1
shows the length of time the various mutants survived
upon exposure to the indicated oxidative stress condition at 20°C. In
the presence of PQ under hyperoxia, the spe-26(hc138) and
clk-1(e2519) mutants survived for a length of time similar
to that of the wild type. In contrast, various alleles of the
daf-2 mutants that display the Age phenotype (6)
at 20°C
(a permissive temperature) and the Daf-c phenotype at 25°C (a
nonpermissive temperature) (4
, 38
) survived for a longer period of time
than the wild type in the presence of PQ under hyperoxia. The
daf-2(e1370) mutant showed a greater resistance to 20 mM
menadione, another intracellular
O2-. generator (39)
, under
hyperoxia than the wild type (Table 1)
. This indicates that the
daf-2 mutants have the Oxr phenotype at a permissive
temperature. Next we examined Oxr of the age-1 mutants. The
age-1(hx546) and age-1(hx524) mutants with
fer-15 background were more resistant to treatment with PQ
under hyperoxia than the fer-15 control mutant (Table 1)
.
This is in accordance with a previous study in which it was shown that
the age-1 mutant was resistant to treatment with PQ (40)
.
Daf-c and NON-Age mutants, such as daf-1,
daf-7, and daf-11, did not display the Oxr
phenotype (Table 1)
, which indicates a correlation between the Age (6
,
12
) and Oxr phenotypes among Daf-c mutants.
Oxr in double mutants
To clarify the effect of other genes on Oxr in the insulin-like
signaling pathway, we measured Oxr in various double mutants of
daf-2(e1370). As shown in Table 1
, a mutation in
daf-16 (m26) and in daf-18(e1375) each completely
suppressed the Oxr phenotype of the daf-2 mutant, although
the sensitivity of the single daf-16 mutant and
daf-18 mutant to oxidative stress was similar to that of the
wild type. This indicates that daf-16 and daf-18
act downstream of daf-2 to confer Oxr as well as Age (6
,
12
). On the other hand, a mutation in the clk-1 gene
significantly enhanced the Oxr phenotype (Table 1)
and the Age
phenotype (16)
in the daf-2(e1370) mutant. This provides
further evidence that a correlation exists between the Age and Oxr
phenotypes in the daf-2 gene network.
Level of sod-3 mRNA in the daf-2
mutants
To examine whether the insulin-like signaling pathway regulates
the expression of genes that encode antioxidant defense enzymes at a
permissive temperature, we measured the level of mRNA transcripts of
SODs and catalase in the daf-2 mutants. The level of
sod-3 mRNA in the daf-2(e1370) and
daf-2(sa189) mutants was significantly higher than that in
the wild type. The levels of mRNA transcripts of sod-1,
sod-2, and catalase (CeCAT) in the daf-2(e1370) and
daf-2(sa189) strains were similar to those in the wild type
(Fig. 1
).
|
The elevated level of sod-3 mRNA in the
daf-2(e1370) mutant was suppressed by the daf-16
(m26) and daf-18 (e1375) mutations (Fig. 2
). The levels of sod-3 mRNA in the age-1(hx542);
fer-15(b26) and age-1(hx546); fer-15(b26) mutants were
higher than that of the fer-15(b26) control mutant (data not
shown). This indicates that the daf-2 genetic pathway does
regulate the expression of the sod-3 gene. Note that
although the single clk-1(e2519) mutant did not have an
elevated level of sod-3 mRNA, the same clk-1
mutation greatly enhanced the level of sod-3 mRNA in the
daf-2 mutant (Fig. 2)
, considering that the clk-1
mutation is known to potentiate the effect of the daf-2
mutation on the Age phenotype (16)
.
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Effect of growth stage on Oxr and sod mRNA level
We investigated whether the growth stage at which animals
are exposed to oxidative stress, affects the Oxr phenotype. Regardless
of growth stage, the wild type did not display the Oxr phenotype when
exposed to oxidative stress (Fig. 3
A). Although the Oxr phenotype was not seen in the
daf-2(e1370) mutant exposed to oxidative stress at the L1
stage, the Oxr phenotype was displayed in daf-2(e1370)
mutant exposed to oxidative stress at and after the L2 stage (Fig. 3A
). Onset of the Oxr phenotype in the daf-2
mutant under growth conditions is coincident with dauer formation in
the wild type under dauer-inducing conditions (8)
.
|
We examined the constitutive level of expression of the
sod-3 gene in the daf-2(e1370) mutant and the
wild type at various developmental stages. sod-3 mRNA
remained constant at a low level in the wild type during its growth
from egg to adult (Fig. 3B
). In contrast, the level of
sod-3 mRNA in the daf-2 mutant increased as it
developed from the egg to the L2 stage, and thereafter remained at this
high level (Fig. 3B
).
Level of sod-3 mRNA in the dauer
To gain an insight into the significance of sod-3
expression, we compared the level of sod mRNA at the dauer stage in
Daf-c mutants that do and do not display the Age phenotype. The
daf-7(e1372) mutant does not display the Oxr phenotype
(Table 1)
, the Age phenotype (6)
, or the elevated level of
sod-3 mRNA at the young adult stage (Fig. 4
), whereas the daf-2(e1370) mutant does display the Oxr and
Age phenotypes as well as an elevated level of sod-3 mRNA at
the young adult stage. On the other hand, the level of sod-3
mRNA in the daf-7 mutant at the dauer stage was as high as
that in the daf-2 mutant at the dauer stage (Fig. 4)
. These
results suggest that the mRNA level of sod-3 is high at the
dauer stage regardless of genetic background and that among Daf genetic
pathways, only the daf-2 pathway regulates sod-3
gene expression at the adult stage.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and Age phenotypes (6)
, but
also the Oxr phenotype. The Oxr phenotype in the daf-2
mutant requires normal daf-16 gene function; this is similar
to other phenotypes of daf-2 mutants (6
, 8
, 12
, 41
42
43
44
). As
daf-2 encodes a member of the insulin receptor family,
defects in an insulin-like signaling pathway regulate oxidative stress
resistance by activating daf-16 gene function. Since
daf-16 encodes an HNF-3/forkhead family transcription factor
(13
, 14
), it can be considered that the daf-2 mutation
activates the transcription of a set of genes involved in oxidative
stress resistance rather than changing the level of activity of already
existing enzymes. Some of these genes should be component(s) of
antioxidant defense systems. We found that the daf-2 mutants
have an elevated level of mRNA transcripts of sod-3 that
encodes Mn-SOD located in the mitochondria, a major site of
O2-. generation. We also found
that a daf-16 and daf-18 mutation each suppressed
both the Oxr phenotype and the increased level of sod-3 mRNA
in the daf-2 mutant. These results suggest that the
insulin-like signaling pathway confers oxidative stress resistance by
regulating the Mn-SOD gene expression.
The biological significance of the relationship between insulin
signaling and Mn-SOD gene expression is not clearly understood. In
vertebrates, insulin is involved in the cellular regulation of glucose
uptake and energy metabolism (45)
. If the insulin-like system in
C. elegans is similar to that in vertebrates, defects in
insulin-like signaling due to a daf-2 mutation may be
responsible for these metabolic changes. In fact, Kimura et al. (4)
pointed out that the protein sequence of the insulin receptor in an
insulin-resistant patient had the same amino acid substitution (46)
as
that in the daf-2(e1391) mutant of C. elegans. In
mammals, the glucose level affects the redox state of the mitochondria
(47)
, which in turn is associated with the level of ROS generation
(48)
. In cultured mouse astrocytes (49)
and human breast carcinoma
cells (50)
, glucose deprivation induces oxidative stress; this suggests
that glucose metabolism affects ROS generation. It has been shown in a
variety of eukaryotes that ROS induce the gene expression of Mn-SOD
(51)
. There is an intriguing link between an insulin-signaling defect
and the gene expression of Mn-SOD in vertebrates. Tumor necrosis factor
, which interferes with insulin-receptor signaling (52)
, induces the
gene expression of Mn-SOD (53)
. The link between insulin signaling and
Mn-SOD could have been conserved among diverse species.
In daf-2 mutants, the Age and Oxr phenotypes and the
elevated level of sod-3 mRNA are closely associated in that
they appear at a permissive temperature at which the Daf-c phenotype is
not displayed (38)
. Moreover, epistasis analysis has shown that
daf-18 acts downstream of daf-2, specifically for
the Oxr phenotype and the increased level of sod-3 mRNA as
well as for the Age phenotype (12)
, whereas daf-18 is not
epistatic to daf-2(e1370) for the Daf phenotype (12
, 41
).
This is additional evidence that supports a close association between
the Age and Oxr phenotypes and the increased level of sod-3
mRNA.
Accumulating evidence suggests the involvement of oxidative
stress in the aging process of C. elegans. In response to
oxidative stress, the level of total SOD activity in young, wild-type
animals increases, whereas this adaptive response diminishes at the
late adult stage (54)
. However, the basal level of total SOD activity
in the wild type does not change as the animals age (28
, 40
). In the
daf-2 (55)
and age-1 (28
, 40
) mutants, the level
of total SOD activity is higher at the late adult stages than the early
adult stage. In a previous study on wild-type C. elegans, we
reported that the life span and aging rate were inversely correlated
with the concentration of environmental oxygen (34
, 56
). The oxidative
stress-sensitive mev-1 mutant has a shorter life span and
faster aging rate than the wild type (34
, 57
, 58
). Hartman et al. (59)
also demonstrated an inverse correlation between life span and
susceptibility to oxidative stress in recombinant inbred strains. These
findings suggest that oxidative damage is an important causative factor
in the aging process of C. elegans. Kimura et al. (4)
suggested that life extension by the daf-2 mutation may be
induced by a mechanism analogous to life extension in mammals caused by
decreased energy usage induced by caloric restriction. Taken together,
the close correlation between the Age and Oxr phenotypes and the
increased level of sod-3 mRNA in the daf-2 mutants shown in
the present study suggests that life extension in the daf-2
mutant may result from a slowing in the rate of accumulation of
oxidative damage caused by normal metabolism, especially mitochondrial
energy production.
The mechanism responsible for daf-2 mutants having an
extended adult life span may be a dauer subprogram for efficient life
maintenance that uncouples from other aspects of dauer formation at
permissive temperatures induced in the adult stage (6
, 12
). The
mechanism responsible for the Oxr phenotype is a candidate for this
subprogram. Dauer larva is resistant to
H2O2 and has elevated
levels of SOD and catalase activity (60)
. We found that
daf-7, a Daf-c and NON-Age ts mutant, has an
elevated level of sod-3 mRNA at the dauer stage, but does
not have an elevated level of sod-3 mRNA (Fig 4)
, or the Oxr
phenotype (Table 1)
at the adult stage. This result supports the idea
that the efficient life maintenance subprogram, including
sod-3, is incorporated into the dauer formation program in
Daf-c, NON-Age, and Non-Oxr mutants such as the
daf-7 mutant; these mutants in the adult stage would not
display the Age or Oxr phenotypes.
We examined Oxr and the mRNA levels of the sod-3 gene in the
daf-2(e1370) mutant at various developmental stages. The Oxr
phenotype and increased sod-3 expression became apparent as
the daf-2 mutant developed (Fig. 3)
. However, they did not
necessarily occur in parallel in that the sod-3 mRNA level
was higher in the daf-2 mutant at the L1 stage than the wild
type, although the Oxr phenotype was not seen in the daf-2
mutant exposed to oxidative stress at the L1 stage. It is possible that
increased sod-3 expression is not sufficient for the Oxr
phenotype. Alternatively, one can suppose that a threshold level of
sod-3 expression is necessary to display the Oxr phenotype
or that the translation product of sod-3 increases later
than the increase in mRNA. More investigations are needed to clarify
these points. The developmental stage at which the daf-2
mutant first displays the Oxr phenotype (Fig. 3A
), the stage
at which the sod-3 mRNA reaches its maximum level in the
daf-2 mutant under growth conditions (Fig. 3B
),
and the stage at which the dauer forms in the wild type under
dauer-inducing conditions (8)
all occur at approximately the L2 stage.
We hypothesize that only the efficient life maintenance mechanism,
including the antioxidant defense mechanism, switches on at the L2
stage in daf-2 mutants kept at a permissive temperature in a
manner similar to that in which all dauer formation programs switch on
under dauer-inducing signals. Furthermore, the efficient life
maintenance mechanism may continue to function in the daf-2
mutants to result in adult life extension.
It is interesting that C. elegans has two Mn-SOD genes,
sod-2 and sod-3, since other eukaryotes are known
to have one (31)
. sod-2 is expressed at almost the same
level in both the adult and dauer stages, whereas sod-3 is
expressed specifically in the dauer stage of NON-Age worms
(Fig. 4)
. Along with the above results, we can suppose that
sod-3 comprises part of the efficient life maintenance
subprogram and switches on at the dauer stage. It may be possible that
sod-2 has an antioxidant function under normal conditions
defining the wild-type adult life span and that sod-3 could
represent another life span-determining program usually executed at the
dauer stage.
The other Age mutant, clk-1(e2519), did not display the Oxr
phenotype (Table 1)
. It is interesting that the clk-1
mutation significantly enhanced the Oxr phenotype (Table 1)
, as well as
the level of sod-3 mRNA (Fig. 2)
, in the
daf-2(e1370) mutant considering that the clk-1
and daf-2 mutations synergistically extend adult life span
(16)
. These results suggest that the clk-1 mutation
potentiates the Age phenotype in the daf-2 mutant through
the same mechanism that regulates the Oxr phenotype and
sod-3 mRNA levels. This means that clk-1 is
involved in longevity in two ways: clk-1 composes the
original clk-1 longevity program (16)
and also the
daf-2 longevity program. clk-1 encodes a
homologue of yeast CAT5/COQ7 (17)
that is involved in gluconeogenesis
(18)
and the synthesis of ubiquinone in the mitochondria (19)
.
Therefore, clk-1 gene function may regulate cellular energy
metabolism.
In our model (Fig. 5
), the defect in insulin-like signaling caused by the daf-2
mutation activates the regulatory system of Mn-SOD gene expression.
This system may be regulated by the level of cellular energy
metabolism, e.g., glucose concentration or mitochondrial redox level,
etc., using the analogy that in mammals the glucose level affects the
regulation of the expression of various genes by insulin (61)
. We can
speculate that the synergistic action of the clk-1 and
daf-2 mutations on life extension may be executed through
the pathway in which changes in mitochondrial energy metabolism induced
by the clk-1 mutation affect the regulatory system of Mn-SOD
gene expression. Further studies are needed to confirm this model. The
present study may provide a simple model system for examining how the
gene expression of SODs is regulated by cellular metabolism and the
endocrine system, and how these are involved in determining
longevity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication October 21, 1998.
Revision received February 22, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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function. Nature (London) 389,610-614[Medline]
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 S. T. Henderson, M. Bonafe, and T. E. Johnson daf-16 Protects the Nematode Caenorhabditis elegans During Food Deprivation. J. Gerontol. A Biol. Sci. Med. Sci., May 1, 2006; 61(5): 444 - 460. [Abstract] [Full Text] [PDF]
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 H. Murakami, K. Bessinger, J. Hellmann, and S. Murakami Aging-Dependent and -Independent Modulation of Associative Learning Behavior by Insulin/Insulin-Like Growth Factor-1 Signal in Caenorhabditis elegans J. Neurosci., November 23, 2005; 25(47): 10894 - 10904. [Abstract] [Full Text] [PDF]
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