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INTRODUCTION |
Two different Na+-coupled dicarboxylate transporters
(NaDC)1 have been identified
in mammalian tissues (1-7). These are NaDC1 and NaDC3. NaDC1 is
Na+-coupled, electrogenic, and exhibits low affinity for
its dicarboxylate substrates. The Kt value
(Michaelis-Menten constant) is in the range of 0.1-4.0 mM
(1-4). This isoform is expressed primarily in the brush border
membrane of intestinal and renal epithelial cells. The physiological
function of NaDC1 is to absorb the intermediates of the citric acid
cycle, such as citrate, succinate,
-ketoglutarate, fumarate, and
malate, in the intestine and kidney. NaDC3 is also a
Na+-coupled and electrogenic dicarboxylate transporter, but
it exhibits relatively higher affinity for its substrates compared with
NaDC1 (5-7). The Kt value is in micromolar range.
The NaDC3 is expressed primarily in the basolateral membrane of
intestinal and renal epithelial cells. However, it is also found in
tissues such as liver, placenta, and brain. NaDC3 in the kidney is
involved in generating the driving force for the organic anion
transporter OAT1 to facilitate the active entry of organic anions into
the tubular cells across the basolateral membrane (8). In the brain, NaDC3 mediates the cellular uptake of N-acetylaspartate, a
process closely linked to myelination (9). Therefore, the physiological functions of the NaDCs may extend beyond the mediation of cellular entry of citric acid cycle intermediates. Recently, we reported on the
molecular identification of the third member of this family in mammals
(10, 11). This transporter, known as Na+-coupled citrate
transporter (NaCT), mediates the cellular uptake of citrate in a
Na+-coupled manner.
In a recent study by Rogina et al. (12), a NaDC-like
transporter, coded by the Indy (for I am
not dead yet) gene, has been
implicated in the regulation of life span in Drosophila. The
investigators of this study suggested that defects in one copy of the
Indy gene (heterozygosity) can lead to less efficient production of cellular energy and that, as a consequence, the metabolic
profile of the fruit fly changes resulting in life span extension. The
eating behavior of the organism is not altered, however. The decreased
generation of cellular energy due to the heterozygous mutation in the
Indy gene creates a biological situation resembling that of
caloric restriction, which in other animal models leads to an extension
of life span (13). Recently, we have identified (14) the transport
function of Drosophila INDY. This transporter mediates the
cellular uptake of a broad spectrum of citric acid cycle intermediates
in a Na+-independent manner. These characteristics of
drINDY have now been confirmed independently by Knauf et al.
(15).
Studies of life span extension are difficult, if not impossible, to
conduct in mammals, particularly in humans. But it is relatively a
simple task to monitor the mean and maximum life span in other animal
models such as Caenorhabditis elegans. A number of features
make C. elegans especially suitable for studies of life span
extension. This organism has a short life span with a mean life span of
~15 days. In addition, there are techniques available to silence
genes in this organism as a means of assessing the role of specific
genes in the maintenance of life span. Therefore, with an aim to
investigate the potential role of NaDC family in life span, we set out
to clone the C. elegans counterparts of mammalian NaDCs
and subsequently to monitor the influence of these transporters on life
span by using the RNAi technique to down-regulate their function. These
studies have successfully led to the molecular and functional
identification of two different Na+-coupled dicarboxylate
transporters (ceNaDC1 and ceNaDC2) analogous to the mammalian NaDC1 and
NaDC3. In addition, studies of the influence of these two transporters
on life span have shown that disruption of the function of the high
affinity transporter ceNaDC2, but not that of the low affinity
transporter ceNaDC1, leads to a significant extension of the average
life span in C. elegans.
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EXPERIMENTAL PROCEDURES |
Nematode Culture--
A wild type nematode strain, C. elegans N2 (Bristol-Myers Squibb Co.), was obtained from the
Caenorhabditis Genetics Center (St. Paul, MN). Nematode
culture was carried out using a standard procedure with a large scale
liquid cultivation protocol (16-19). The nematodes were cleaned by
sedimentation through 15% (w/v) Ficoll 400 in 0.1 M NaCl.
The pellet was then used for total RNA preparation.
Extraction and Purification of Poly(A)+
RNA--
Total RNA was isolated using the TRIzol reagent (Invitrogen).
Poly(A)+ mRNA was purified by affinity chromatography
using oligo(dT)-cellulose.
Reverse Transcription (RT)-PCR and Hybridization Probe
Preparation--
A pair of PCR primers specific for the putative
C. elegans nadc1 gene was designed based on the sequence of
the cosmid F31F6.6 (GenBankTM accession number Z69884),
5'-GCC TCC AAG CAA AAT GTC TC-3' (forward primer) and 5'-CTA ACG CAA
ATC CAC CTC C-3' (reverse primer). A second pair of PCR primers
specific for putative C. elegans nadc2 gene was designed
based on the sequence of the cosmid K08E5.2 (GenBankTM
accession number Z30974), 5'-TCA TCC TTC CAA CAC CAT CC-3' (forward
primer) and 5'-ACC ATT CCA CTT CCA AAC AC-3' (reverse primer).
Poly(A)+ RNA (~0.5 µg) isolated from mixed stage
C. elegans was taken as template to perform RT-PCR using an
RT-PCR kit from PerkinElmer Life Sciences. A single RT-PCR product was
obtained with an estimated size of ~1.0 and ~0.9 kb for the
cenadc1 and the cenadc2 genes, respectively, as
predicted by the distance between the two primers in each pair. The
RT-PCR products were gel-purified and subcloned into pGEM-T Easy Vector
(Promega, Madison, WI). The molecular identity of the inserts was
established by sequencing. These cDNA fragments were used as probes
to screen a C. elegans cDNA library.
Construction of a Directional C. elegans cDNA
Library--
The SuperScript Plasmid System from Invitrogen was used
to establish the cDNA library using the poly(A)+ RNA
from C. elegans. The transformation of the ligated cDNA
into Escherichia coli was performed by electroporation using
ElectroMAX DH10B competent cells. The bacteria plating, the filter
lifting, the DNA fragment labeling, and the hybridization methods
followed the routine procedure (20). The DNA sequencing of the
full-length ceNaDC1 cDNA and ceNaDC2 cDNA clones was performed
using an automated PerkinElmer Life Sciences Applied Biosystems 377 Prism DNA sequencer and the Taq DyeDeoxy terminator cycle
sequencing protocol.
Vaccinia/T7 Expression System--
Functional expression of the
ceNaDC cDNAs in mammalian cells was done in human retinal pigment
epithelial (HRPE) cells using the vaccinia virus expression system as
described previously (5, 7, 9). HRPE cells grown in 24-well plates were
infected with a recombinant vaccinia virus (VTF7-3) at a
multiplicity of 10 plaque-forming units/cell. The virus was allowed to
adsorb for 30 min at 37 °C with gentle shaking of the plate. Cells
were then transfected with the plasmid DNA (empty vector pSPORT or ceNaDC1 cDNA or ceNaDC2 cDNA constructs) using the lipofection procedure (Invitrogen). The cells were incubated at 37 °C for 12 h and then used for determination of transport activity. Cells transfected with pSPORT alone without the cDNA insert were used as
the control to determine endogenous transport activity in these cells.
[3H]Succinate uptake was determined at 37 °C as
described previously (5, 7, 9). In most experiments, the uptake medium
was 25 mM Hepes/Tris (pH 7.5), containing 140 mM NaCl, 5.4 mM KCl, 1.8 mM
CaCl2, 0.8 mM MgSO4, and 5 mM glucose. In experiments in which the cation and anion
dependence of the transport process was investigated, NaCl was replaced
iso-osmotically by LiCl, KCl, sodium gluconate, or
N-methyl-D-glucamine (NMDG) chloride. The transport activity in cDNA-transfected cells was adjusted for the
endogenous activity to calculate the ceNaDC cDNA-specific activity.
The endogenous succinate transport activity in vector-transfected cells
was always less than 10% compared with the succinate transport activity measured in cells transfected with either ceNaDC1 cDNA or
ceNaDC2 cDNA. Experiments were done in triplicate, and each experiment was repeated at least three times. Results are presented as
means ± S.E.
Semi-quantitative RT-PCR--
An RT-PCR assay with the
cenadc1- or cenadc2-specific primers described
above was used to study the developmental stage-specific expression
pattern of ceNaDC1 mRNA and ceNaDC2 mRNA. A Quantum RNA 18 S
internal standard (Ambion, Austin, TX) was used for the semi-quantitative RT-PCR. Total RNA (~1.0 µg) isolated from
different developmental stages of C. elegans (embryo, early
larva, late larva, and adult) was taken as template to perform reverse
transcription using an RT-PCR kit from PerkinElmer Life Sciences. The
reverse transcription was initiated with random hexamers and carried
out in a DNA thermal cycler (GeneAmp PCR System 9600) and thin-walled reaction tubes (PerkinElmer Life Sciences) at 42 °C for 60 min, followed by incubation at 99 °C for 5 min to inactivate the reverse transcriptase (Maloney murine leukemia virus-reverse transcriptase). Reverse transcription was followed by PCR in a multiplex format, in
which the gene-specific primers and the primers for the internal control (18 S rRNA) with their competimers were combined at a predefined ratio. The PCR cycle number was titrated according to the
manufacturer's protocol to ensure that the reaction was within the
linear range. A competimer was included to prevent the highly abundant
rRNA from being overwhelmingly amplified during the reaction, and an
optimal 18 S primer:competimer ratio was also pre-established by trial
and error. The resultant multiplex PCR products were resolved in an
1.0% agarose gel, and the intensity of the gene-specific and the 18 S
rRNA-specific bands was determined using SpectraImager 5000 Imaging
system and AlphaEase 32-bit software (Alpha Innotech, San Leandro, CA).
The steady state levels of ceNaDC1 mRNA and ceNaDC2 mRNA at
different developmental stages were assessed from the relative ratios
of the intensity of the ceNaDC1-specific RT-PCR product or
ceNaDC2-specific RT-PCR product to the intensity of the 18 S
rRNA-specific RT-PCR product at each of these stages.
Analysis of Tissue-specific Expression Pattern of cenadc1 and
cenadc2--
To study the tissue-specific expression pattern of the
nadc1 and nadc2 genes in C. elegans,
transcriptional cenadc1::gfp and cenadc2::gfp fusion genes were constructed,
and transgenic animals expressing these transgenes were developed. The
expression pattern of the cenadc1 and cenadc2
genes was investigated in live transgenic animals based on the
expression pattern of the GFP reporter. A pair of primers for
construction of a transcriptional
cenadc1::gfp fusion gene was designed
to amplify the cenadc1 promoter. The forward primer, 5'-CGC
GTC GAC GCT TAC ATC ATT CTT GTA TTT TTC-3', corresponds to the
nucleotide positions 28,630-28,659 of the cosmid F31F6
(GenBankTM accession number Z69884). The primer contains an
incorporated SalI restriction site at its 5' end. The
reverse primer, 5'-ATA GGA TCC ATG ATT GGA GGC TCT GCA ATA CTA-3',
corresponds to the nucleotide positions 29,797-29,772 in the same
cosmid. A BamHI site was incorporated in this primer at the
5' end. The SalI and BamHI sites were introduced
into these primers for subsequent cloning into a GFP expression vector.
An ~1.2-kb DNA fragment of the cenadc1 promoter was
amplified using the cosmid F31F6 DNA as template. Similarly, a pair of
primers for construction of a transcriptional
cenadc2::gfp fusion gene was also
designed. The forward primer, 5'-GTC GAC AAA ATA TGT ATT AGC CAC ATA
AAA CCC-3', corresponds to the nucleotide positions 13,966-13,998 of
the cosmid K08E5 (GenBankTM accession number Z30974). The
reverse primer, 5'-GGA TCC ATT TTC CGC ACA TGC CGA ATT TGC AT-3',
corresponds to the nucleotide positions 15,461-15,432 in the same
cosmid. A SalI site and a BamHI site were
incorporated in these primers for subsequent subcloning purposes. A
~1.5-kb DNA fragment of the cenadc2 promoter was amplified using the cosmid K08E5 as template. The PCR products were digested with
SalI and BamHI and inserted into a GFP expression
vector pPD117.01 (a generous gift from Dr. A. Fire, Carnegie
Institution, Baltimore, MD) at a SalI/BamHI site.
In these minigene constructs, a built-in mec7 promoter
(~0.9 kb) in the expression vector was replaced by the
cenadc1 and cenadc2 promoter fragments in such a
way that the GFP transcription is under control of the putative promoters of the cenadc1 and cenadc2 genes,
respectively. The minigene fusion constructs were verified by sequence
analysis. Transgenic lines were established using a standard germ line
transformation protocol (17, 18). Syncytial gonad injection was carried
out according to a standard procedure (18). For microinjection, a
computerized injection system, Transjector 5246 and
Micromanipulator 5171 from Eppendorf (Hamburg, Germany), and a Nikon
Eclipse TE 300 inverted microscope with Nomarski differential
interference contrast optics were used. A cloned mutant collagen gene
containing the rol-6 (plasmid pRF4, kindly provided by Dr.
M. Koelle, Yale University School of Medicine, New Haven, CT) was used
as a dominant genetic marker for DNA transformation. Coinjection of
this dominant marker with the GFP fusion constructs allowed progeny
selection of the transformed animals by their "roller" phenotype.
The F1 rollers were picked up according to their characteristic rolling behavior and cultured individually to establish transformed lines. F2
rollers with extrachromosome arrays were selected for fluorescence microscopy to determine the GFP expression pattern. Stable transgenic lines were established by the
-irradiation method from the F2 rollers, and the background was cleaned up by several times of outcross
(17, 21).
Double Labeling Fluorescent Protein Expression System--
Two
modified versions of the Aequora victoria green fluorescent
protein (GFP), designated as CFP and YFP with cyan-shifted and
yellow-shifted spectra (22), respectively, were used to simultaneously
follow the expression patterns of ceNaDC1 and ceNaDC2 in C. elegans. For the construction of the cenadc1
promoter-driven CFP and the cenadc2 promoter-driven YFP
expression vectors, the GFP coding region in the GFP-expression vector
cenadc1::gfp (pPD117) and
cenadc2::gfp (pPD117) was substituted
by the CFP and YFP coding regions, respectively. The CFP and YFP coding
regions (~950 bp) were obtained by an
EcoRI/KpnI digestion of the vectors L4666 (pPD133.58) and L4664 (pPD133.51), respectively (kindly provided by Dr.
A. Fire, Carnegie Institution, Baltimore, MD). The
cenadc1::cfp and
cenadc2::yfp expression vectors were
linearized by SalI digestion and coinjected into the distal
arms of the C. elegans syncytial gonads as described
earlier. The extrachromosome arrays were used for fluorescence
microscopy to compare the expression pattern of CFP and YFP in the same
transgenic animal. Epi-fluorescence microscopic analysis of the
expression of CFP and YFP was performed using an Axiophot microscope
(Carl Zeiss, Thornwood, NY). Excitation and emission filter settings
are as follows: for CFP examination, excitation at 436 ± 20 nm,
dichroic 455 nm LP, and emission at 480 ± 40 nm; for YFP
examination, excitation at 500 ± 20 nm, dichroic 515 nm LP, and
emission at 535 ± 30 nm LP (22). The filter sets were purchased
from Chroma Technology (Brattleboro, VT). The SPOT-cooled CCD color
digital camera (Diagnostic Instruments Inc., St. Sterling Heights, MI)
and its associated data acquisition software were used to record the
fluorescence images.
Bacteria-mediated RNA Interference (RNAi)--
A fragment of
the coding region of ceNaDC1 cDNA was generated by PCR and
subcloned into a pGEM-T easy vector (Promega, Madison, WI). The DNA
fragment was released by EcoRI digestion and inserted into a
"double T7" plasmid (pPD129.36, a generous gift from Dr. A. Fire,
Carnegie Institution, Baltimore, MD) at an EcoRI site within
the multiple cloning site. A host strain DH5
was used for the first
transformation. Competent host bacteria HT115 (DE3) (kindly provided by
the Caenorhabditis Genetics Center, St. Paul, MN))
expressing T7-RNA polymerase from an inducible promoter was prepared
using a standard CaCl2 method (20). The double T7
promoter-containing plasmid with the cenadc1 gene-specific
DNA fragment inserted between the two T7 promoter regions was
transformed into the competent HT115 (DE3) cells and plated onto
standard LB + tetracycline (12.5 µg/ml) + ampicillin (100 µg/ml)
plates. HT115 cells harboring the double-T7 plasmid were cultured and
induced to express dsRNA using 0.4 mM
isopropyl-
-D-1-thiogalactopyranoside at 37 °C for 4 h. The experimental worms were transferred onto
isopropyl-
-D-1-thiogalactopyranoside-containing nematode
growth medium plates with the induced bacteria HT115 lawn for bacteria
feeding experiments. The empty vector pPD129 was processed similarly
for use as a negative control, and the bacteria harboring this plasmid
were used to feed the control worms. Sufficient quantities of bacteria
HT115 were seeded on the testing plates for the worms to consume to
prevent the worms from starving and to ensure that dsRNA was always
present in the testing plates during the entire experimental period for
the experimental worms. A similar experimental strategy was used for
ceNaDC2. To serve as a positive control for the bacteria-mediated RNAi
in the assessment of the influence of ceNaDC1 and ceNaDC2 on life span,
we monitored the influence of DAF-2 on life span using an identical
experimental approach. Homozygous daf-2
/
knockout in C. elegans is known to enhance the life span
dramatically (~2-fold) (23, 24). Therefore, if the knockdown of DAF-2
function by bacteria-mediated RNAi in C. elegans doubles the
life span, this can be taken as a positive control for the validity of
the experimental approach to assess the role of ceNaDC1 and ceNaDC2 in
life span. For this purpose, we obtained an ~0.8-kb DNA fragment specific for C. elegans DAF-2 by RT-PCR using the following
primer pairs: 5'-CGAACAAAACACATCACAGAC-3' (forward primer) and
5'-TCCATCATTTCCATCACAACC-3' (reverse primer) using the nematode total
RNA as the template. This fragment was then subcloned into pGEM-T easy
vector. The fragment was then released from the vector by
EcoRI digestion, and the released insert was cloned into the
double T7 plasmid pPD129.36 at the EcoRI site at the
multiple cloning region. Shuttling of this plasmid into HT115 (DE3)
bacteria, induction of double-stranded RNA, and feeding of the worms
with the bacteria were carried out as described earlier.
Life Span Measurement--
Life span of age-synchronous
nematodes was determined at 20 °C. Eggs obtained from gravid
hermaphrodites using an alkaline hypochlorite treatment procedure (18)
were dispensed on nematode growth medium Petri dishes with bacteria
lawn and allowed to hatch. Worms were inspected every day until death
and were scored as dead when they were no longer able to move even in
response to prodding with a platinum-wire pick. Each day, dead worms
were removed from plates and the deaths were recorded. Experiments were
started with 60 worms for each RNAi treatment (10 per plate). The worms
were transferred to a new plate every day during the reproductive
period and every 3 days afterward to avoid contamination by their
offspring. Worms that died from matricidal hatching (the bag-of-worms
phenotype) and the worms that crawled off the plates or burrowed into
the agar were replaced by spare worms. A backup reservoir plate of
~30 spare worms was started at the same time as the experimental
worms and was identically treated for this purpose. To avoid the
influence of any potential subjective judgment of the experimenter in
identifying the dead worms on the experimental outcome, the life span
measurement studies were repeated with the experimenter blinded with
regard to the identity of the individual experimental groups.
Statistical analysis was performed using the Microsoft EXCEL 2000 analysis ToolPak. Mean life spans from different groups were compared
using the Student's t test assuming unequal population variances.
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RESULTS |
Molecular Cloning and Structural Characterization of ceNaDC1 and
ceNaDC2--
The cloned ceNaDC1 cDNA is 1,989 bp long and contains
a poly(A) tail (GenBankTM accession number AY090484). The
5'- and 3'-untranslated regions of this cDNA are 12 and 173 bp
long, respectively. The ceNaDC1 protein, deduced from the coding region
of the cDNA, contains 582 amino acids (Fig.
1) with a predicted molecular mass of 64 kDa and an isoelectric point of 6.64. The ceNaDC2 cDNA is 2,250 bp
long and contains a poly(A) tail (GenBankTM accession
number AY090485). The 5'- and 3'-untranslated regions of this cDNA
are 50 and 500 bp long, respectively. The ceNaDC2 protein, deduced from
the coding region of the cDNA, contains 566 amino acids (Fig. 1)
with a predicted molecular mass of 62 kDa and an isoelectric point of
7.69. According to the Kyte-Doolittle plot with a 21-amino acid window
size, ceNaDC1 as well as ceNaDC2 possess 12 putative transmembrane
domains.

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Fig. 1.
A multiple protein sequence comparison of the
representative members in the NaDC superfamily. The software
PILEUP (version 10.2) in the GCG package (from the Genetic Computer
Group Inc., Madison, WI) was used to establish this multiple sequence
alignment. Gaps are introduced to make an optimum alignment
and are indicated by dots/dashes. The
names of the transporters are indicated at the beginning of the protein
sequence. The sodium/symporter family signature sequence is highlighted
by a box.
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Following a multiple protein sequence alignment of the two ceNaDCs, the
three members of the human NaDC family, and the drINDY using the PILEUP
and in combination with the MOTIFS program in the GCG package, a sodium
symporter family signature motif was identified within these
transporter proteins (Fig. 1). A consensus pattern established for the
signature sequence is as follows: (S)SX(2)FX(2)P(V)(G)X(3)NX(I)V,
where the X denotes the flexible amino acid residues
preceding the number in parentheses, and the numerical value indicates
the permitted number of the flexible amino acid residues in the
consensus. This sodium symporter family is a group of integral membrane
proteins that mediate the cellular uptake of a wide variety of
molecules including di- or tricarboxylates and sulfate by a transport
mechanism involving sodium cotransport (sodium symporters). They are
grouped into a single gene family on the basis of sequence and
functional similarities. This group consists of the following proteins:
the sodium/sulfate cotransporters and sodium/dicarboxylate
cotransporters identified in yeast, C. elegans,
Drosophila, and mammals; the putative sulfur
deprivation response regulator (SAC1) from Chlamydomonas
reinhardtii; and the hypothetical protein YfbS from
E. coli (25). These transporter proteins usually consist of
430-620 amino acids. They are highly hydrophobic and contain 11 or 12 putative transmembrane regions. The highly conserved sodium symporter
signature motif is located in or near the penultimate transmembrane domain.
The molecular identity of mammalian or C. elegans functional
counterpart of drINDY is not known at present. Therefore, we compared
the primary structure of ceNaDC1 and ceNaDC2 with that of drINDY and
mammalian NaDC1, NaDC3, and NaCT. With a pairwise comparison analysis,
ceNaDC1 is more closely related to drINDY (51% similarity and 37%
identity) than ceNaDC2 (46% similarity and 35% identity). Similarly,
hNaDC1 and hNaCT are more closely related to drINDY (52% similarity
and 40% identity) than hNaDC3 (50% similarity and 37% identity).
However, the differences are small, and it is difficult to conclude
whether ceNaDC1 or ceNaDC2 is the C. elegans functional
counterpart of drINDY based on the structural comparison. Similarly,
this structural analysis does not allow definitive conclusion with
regard to the question of whether NaDC1or NaDC3 is the mammalian
functional counterpart of drINDY. Structural comparison reveals that
both ceNaDCs have similar sequence homology with hNaDC1, hNaDC3, and
hNaCT. Thus, the sequence data have failed to provide any useful hint
with respect to the functional identity of the two ceNaDCs.
The cenadc1 and cenadc2 genes are located on
chromosomes X and III, respectively. Both genes, excluding the
unidentified promoter region in respective genes, are ~3.5 kbp in
size (C. elegans data base, ACeDB, data version WS57). The
presence of 10 exons in the cenadc1 gene and 8 exons in
cenadc2 gene was deduced by a comparison between the
sequences of the cloned cDNAs and the respective genes in the
GenBankTM data base (F31F6.6 and K08E5.2) from the nematode
genome sequence project. The structural organization of the
cenadc1 and cenadc2 genes is shown in Fig.
2.

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Fig. 2.
Structure of cenadc1 and
cenadc2 genes. Exons are indicated by
filled boxes and numbered accordingly; introns are shown by
solid lines. The untranslated regions in exons are indicated
by open boxes. The consensus polyadenylation signal AATAAA
is also shown. Sizes and positions of the exons and introns are drawn
to the exact scale.
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Functional Characterization of ceNaDC1 and ceNaDC2 Using a
Heterologous Expression System--
The functional analysis of the
cloned ceNaDCs was carried out by heterologous expression of the
cDNAs in HRPE cells using the vaccinia virus expression system (5,
7, 9). Cells transfected with vector alone served as the control. The
transport function was monitored by the uptake of
[3H]succinate. Initial studies on the time course of
uptake indicated that the uptake was linear at least up to 5 min. All
subsequent studies were therefore carried out with a 2-min incubation.
With a succinate concentration of 10 µM and in the
presence of Na+, the uptake of succinate increased 12-fold
in cells expressing ceNaDC1 compared with control cells (Fig.
3A). Under similar conditions, the increase in succinate uptake was 24-fold in the case of ceNaDC2. Thus, both ceNaDC1 and ceNaDC2 mediate the uptake of succinate in the
presence of Na+. The uptake via these two transporters was,
however, obligatorily dependent on the presence of Na+
because substitution of Na+ with Li+,
K+, or NMDG abolished completely the cDNA-induced
increase in succinate uptake. There was no involvement of anions in the
uptake process as indicated by comparable uptake activities for both
transporters in the presence of NaCl or sodium gluconate (data not
shown).

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Fig. 3.
A, ion dependence of ceNaDC-mediated
succinate uptake in HRPE cells. Uptake of 10 µM succinate
was measured in buffers containing 140 mM concentrations of
sodium, lithium, potassium, or NMDG (as chloride salts). Values
represent means ± S.E. for four determinations. Uptake of 10 µM succinate measured in the vector (pSPORT)-transfected
cells served as a control for endogenous uptake activity. The uptake in
cDNA-transfected cells is given as percent of uptake in
vector-transfected cells. B, substrate specificity of the
ceNaDC-mediated uptake. Uptake of 10 µM
[3H]succinate was measured in the absence or presence of
potential inhibitors (5 mM) in cells transfected with
vector alone, ceNaDC1 cDNA, or ceNaDC2 cDNA. The
cDNA-specific uptake was calculated by adjusting for the uptake in
vector-transfected cells. The cDNA-specific uptake in the absence
of inhibitors was taken as the control (100%), and the uptake in the
presence of inhibitors is given as percent of this control value.
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The substrate selectivity of the uptake process mediated by ceNaDCs was
then studied by competition analysis by monitoring the ability of
various monocarboxylates and dicarboxylates (5 mM) to
compete with succinate for the uptake process (Fig. 3B). Uptake measurements were made in parallel in vector-transfected cells
and in cDNA-transfected cells, and then the cDNA-specific uptake was calculated by subtracting the uptake in vector-transfected cells from the uptake in cDNA-transfected cells. Only the
cDNA-specific uptake was used in the analysis. Among the various
dicarboxylates tested, the ceNaDC1-mediated succinate uptake was
inhibited markedly by fumarate, malate,
-ketoglutarate,
dimethylsuccinate, and N-acetylaspartate. In contrast to
fumarate, its stereoisomer maleate failed to compete with succinate for
transport via ceNaDC1. Malonate, a structural homolog of succinate, not
only failed to inhibit the uptake of succinate but actually caused a
significant stimulation of succinate uptake. The monocarboxylates
pyruvate, lactate, and
-hydroxybutyrate caused only a minimal
inhibition of succinate uptake.
The substrate selectivity of ceNaDC2 was more or less similar to that
of ceNaDC1. The uptake of succinate mediated by ceNaDC2 was inhibited
significantly by fumarate, malate,
-ketoglutarate, dimethylsuccinate, and N-acetylaspartate, whereas the
monocarboxylates had only a minimal effect. However, there were some
notable differences between ceNaDC1 and ceNaDC2. Maleate was able to
inhibit ceNaDC2-mediated succinate uptake, whereas ceNaDC1-mediated
succinate uptake was not affected. Malonate, which caused a significant
stimulation of succinate uptake via ceNaDC1, had minimal effect on
succinate uptake via ceNaDC2. Another notable feature was that fumarate and malate were much more potent in inhibiting ceNaDC2-mediated succinate uptake than in inhibiting ceNaDC1-mediated succinate uptake,
suggesting that there may be significant differences in substrate
affinities between the two transporters. But, interestingly the trend
in the inhibitory potency was opposite for dimethylsuccinate and
N-methylaspartate. These two dicarboxylate derivatives were more potent in inhibiting ceNaDC1-mediated succinate uptake than in
inhibiting ceNaDC2-mediated succinate uptake.
The cDNA-specific succinate uptake was saturable for ceNaDC1 as
well as for ceNaDC2, and the data conformed to the Michaelis-Menten equation describing a single saturable system (data not shown). The
Michaelis-Menten constant (Kt) was 0.73 ± 0.05 mM for ceNaDC1 and 60 ± 9 µM for
ceNaDC2. Thus, with succinate as the substrate, ceNaDC2 exhibits a
10-fold greater affinity than ceINDY1. The competition studies suggest
that the same may be true for other dicarboxylate substrates such as
fumarate and malate. These data show that ceNaDC1 is a low affinity
Na+/succinate cotransporter, and ceNaDC2 is a high affinity
Na+/succinate cotransporter. Therefore, these two C. elegans dicarboxylate transporters correspond at the functional
level to mammalian NaDC1 and NaDC3, respectively. NaCT shows very
little ability to transport succinate and thus is not related to either
ceNaDC1 or ceNaDC2 in terms of transport function.
Drosophila INDY does have the ability to transport various
dicarboxylate intermediates of citric acid cycle (14, 15). But the
transport function is not Na+-dependent.
Furthermore, Drosophila INDY has a much higher affinity for
citrate, a tricarboxylate, than for dicarboxylates (14).
The effect of Na+ on the uptake of succinate was then
investigated by measuring the uptake in the presence of varying
concentrations of extracellular Na+ in cells transfected
with either ceNaDC1 cDNA or ceNaDC2 cDNA. Again, the uptake
values were adjusted for the endogenous uptake activity measured under
identical conditions in cells transfected with vector alone. The
concentration of Na+ in the uptake medium was varied from 0 to 140 mM. The osmolality of the medium was maintained by
adding appropriate concentrations of NMDG chloride as a substitute for
NaCl. The relationship between the cDNA-specific uptake and
Na+ concentration was sigmoidal for both ceNaDC1 and
ceNaDC2, suggesting the involvement of multiple Na+ ions
per succinate molecule transported. The uptake rates failed to reach
saturation within the concentration range of Na+ employed
in these studies.
Developmental Stage-specific Expression Pattern of ceNaDC1 mRNA
and ceNaDC2 mRNA--
To monitor the relative expression levels of
ceNaDC1 mRNA and ceNaDC2 mRNA during different stages of
C. elegans development, synchronized cultures were obtained,
and total RNA was isolated at each of the following four stages of
development: embryo, early larva (larva stages 1 and 2), late larva
(larva stages 3 and 4), and adult. The steady state levels of mRNAs
for ceNaDC1 and ceNaDC2 were then determined by semi-quantitative
RT-PCR with 18 S rRNA as an internal control for variations in RNA
input into RT-PCRs. The levels of ceNaDC1 mRNA and ceNaDC2 mRNA
were compared at different developmental stages based on relative
intensities of ceNaDC-specific RT-PCR products compared with that of
18 S rRNA-specific RT-PCR product (Fig.
4). ceNaDC1 mRNA expression was not
detectable at the embryo stage. Abundant expression of this mRNA
was evident, however, at the early larva stage. There was a transient
decrease in ceNaDC1 mRNA levels at the late larva stage, but the
levels increased again during subsequent development into the adult
stage. The levels of ceNaDC1 mRNA as assessed by the relative band
intensities of RT-PCR products for ceNaDC1 and 18 S rRNA at these four
stages, namely embryo, early larva, late larva, and adult, were 0, 0.87, 0.24, and 0.77. In the case of ceNaDC2, the mRNA was below
detectable levels in the embryo, but the expression was easily
detectable at the early larva stage. The levels of mRNA reached the
maximum at the late larva stage. The relative mRNA levels for
ceNaDC2 at the four stages (embryo, early larva, late larva, and adult) were 0, 2.5, 4.2, and 3.7.

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Fig. 4.
Developmental stage-specific expression of
ceNaDC1 and ceNaDC2. Following RT-PCR, 10 µl of
the products were separated in a 1.2% (w/v) agarose gel to show the
size and intensity of the cenadc gene-specific and the 18 S
rRNA-specific fragments. A 1.0-kb DNA marker (Invitrogen) was used as a
molecular mass standard. The upper bands (~850 bp long) were derived
from the cenadc1 (upper panel) and
cenadc2 (lower panel) transcripts; the lower
bands (~480 bp) were amplified from 18 S rRNA and served as an
internal control. The RNA samples that served as templates were
prepared from different developmental stages of C. elegans:
embryos (Emb), early larva stage (L1&2), late
larva stage (L3&4), and adults. In addition, RNA prepared
from a mixture of C. elegans at different developmental
stages was also used (Mix).
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Tissue-specific Expression Pattern of cenadc1 and cenadc2
Genes--
We first studied the tissue expression pattern of
cenadc1 and cenadc2 genes in C. elegans using the transgenic GFP fusion technique in which the
transgene consisted of the cenadc1 promoter fused with GFP
cDNA or the cenadc2 promoter fused with GFP cDNA. In
both cases, the expression of GFP is controlled by the respective promoter. Thus, the expression pattern of GFP would match the expression pattern of the cenadc1 and cenadc2
genes because of the control of the expression of the GFP reporter by
the respective gene-specific promoters. With this technique, we found
that GFP expression is restricted to the intestinal tract whether the
expression of GFP is driven by the ceNaDC1 promoter or by
the ceNaDC2 promoter (Fig. 5,
A and B), indicating that both cenadc1
and cenadc2 genes are expressed in the intestinal tract. The
expression pattern is evident from the early larva stage through the
adult stage (data not shown). The GFP fluorescence is detectable
throughout the intestinal tract, starting from the pharynx all the way
through the anus. In the case of both promoters, the expression level of GFP is significantly greater in the anterior half of the intestine than in the posterior half.

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Fig. 5.
Tissue-specific expression pattern of
cenadc1 and cenadc2 genes.
Expression of GFP was driven by the cenadc1 (A)
and cenadc2 (B) gene promoters in stably
transformed transgenic C. elegans. The insets A1
and B1 are the bright field images (low magnification) of
the animals for A and B, respectively.
C, (YFP expression driven by the cenadc2
promoter) and D (CFP expression driven by the
cenadc1 promoter) show the expression pattern of YFP and CFP
in the same animal. The inset C1/D1 is the bright field
image of the same worm for both C and D.
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Because both cenadc1 and cenadc2 are expressed in
the same tissue, we employed a double-labeling approach to verify the
coexpression pattern of the two genes. In this approach, we used two
different fluorescent protein reporters, each driven independently by
either cenadc1 promoter (CFP) or cenadc2 promoter
(YFP). Transgenic animals were developed that expressed both of the
reporter constructs. The expression of CFP as well as YFP was then
examined in the same transgenic animal under a fluorescence microscope
with different excitation and emission filter settings. These
experiments showed that the cenadc2 promoter-controlled YFP
and the cenadc1 promoter-controlled CFP were coexpressed in
the intestinal tract (Fig. 5, C and D). This
expression pattern was confirmed with at least 10 transgenic animals.
Influence of RNAi-mediated Knockdown of the Function of ceNaDC1 and
ceNaDC2 on Average Life Span--
The knockdown of the function of
ceNaDC1 by feeding the wild type N2 worms on bacteria expressing the
ceNaDC1-specific dsRNA did not show any significant influence on
average life span nor on the maximal life span (Fig.
6). The average life span of these worms
was same as that of the worms fed on bacteria harboring the empty
vector pPD129 (pPD129 control, 15.3 days; ceNaDC1 knockdown, 14.8 days,
p > 0.05). In contrast, the knockdown of the function of ceNaDC2 by feeding the wild type N2 worms on bacteria expressing the
ceNaDC2-specific dsRNA enhanced significantly (p < 0.0001) the average life span of the worms (pPD129 control, 15.3 days; ceNaDC2 knockdown, 17.6 days). The increase in average life span induced by ceNaDC2 knockdown was 15%. We used DAF-2 knockdown as a
positive control in these experiments. Worms feeding on bacteria expressing DAF-2-specific dsRNA exhibited an average life span of 30 days, showing that the knockdown of the function of DAF-2 doubles the
average life span. This influence of DAF-2 knockdown on life span is
similar to the influence of homozygous knockout of daf-2
gene function on life span (23, 24). This attests to the validity of
the experimental approach indicating that the bacteria-mediated RNAi
strategy is as effective as homozygous knockout strategy.

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Fig. 6.
Influence of the knockdown of ceNaDC1 and
ceNaDC2 on life span in C. elegans. The knockdown
of ceNaDC1 and ceNaDC2 was carried out by feeding the worms with
bacteria producing ceNaDC1- or ceNaDC2-specific dsRNA. The knockdown of
DAF-2 was used as a positive control. The survival curves were plotted
according to the Kaplan-Meier algorithm using Sigma Plot (version 6.0, SPSS Inc., Chicago). These curves show the survival
probability of the wild type animals at a given day after hatching
under the influence of the gene-specific dsRNAs. Each group was from a
total of four experiments. The total number of worms in each
group at the beginning of the life span experiment was 240.
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DISCUSSION |
We have described in this paper the cloning and functional
characterization of two transporters in C. elegans that
mediate the transport of several intermediates of the citric acid
cycle. Both transporters are Na+-coupled and exhibit broad
substrate specificity for dicarboxylates. They do not interact with
monocarboxylates. At the functional level, these two transporters,
named ceNaDC1 and ceNaDC2, resemble the mammalian
Na+-coupled dicarboxylate transporters NaDC1 and NaDC3, respectively.
Even though ceNaDC1 and ceNaDC2 generally resemble NaDC1 and NaDC3,
respectively, in terms of functional characteristics, there is one
important difference. This difference relates to the interaction of
these transporters with certain derivatives of succinate such as
dimethylsuccinate and N-acetylaspartate. Dimethylsuccinate
is considered to be a specific substrate for mammalian high affinity
transporter NaDC3 (26, 27). The low affinity transporter NaDC1 does not
tolerate substitutions in the carbon backbone of succinate. Thus,
dimethylsuccinate and dimercaptosuccinate are recognized
preferentially by NaDC3. In contrast to the mammalian counterparts, it
is ceNaDC1, the low affinity transporter in C. elegans, that
interacts with dimethylsuccinate with much higher affinity compared
with ceNaDC2. Interaction with N-acetylaspartate also
follows a similar pattern. In mammals, NaDC3 shows high affinity for
this succinate analog (9). In contrast, it is ceNaDC1, not ceNaDC2,
that shows high affinity for this compound.
Na+-activation kinetics of succinate uptake mediated by
ceNaDC1 and ceNaDC2 shows that multiple Na+ ions are
involved in the transport mechanism. Succinate exists as a divalent
anion under the experimental conditions (i.e. pH 7.5), and
therefore the number of Na+ ions involved per transport
cycle will determine whether or not the transport process is influenced
by membrane potential. However, the exact number of Na+
ions transported with succinate per transport cycle could not be
determined in the present studies because the activation of succinate
uptake by Na+ did not saturate within the range of
Na+ concentrations employed in the study. We tried to
express ceNaDC1 and ceNaDC2 in Xenopus laevis oocytes to
evaluate the electrogenic nature of these two transporters by using the
two-microelectrode voltage clamp method, but the transporters were not
functionally expressed in this heterologous system. We do not know the
reasons for the lack of expression. We are currently trying different expression vectors for this purpose. Successful expression of these
transporters in X. laevis oocytes may become essential to demonstrate unequivocally whether or not ceNaDCs are electrogenic.
In mammals, the expression of NaDC1 is restricted primarily to the
intestine and kidney, whereas the expression of NaDC3 is evident not
only in the intestine and kidney but also in the liver, brain, and
placenta (26). Furthermore, NaDC1 and NaDC3 exhibit differential
distribution in the apical versus basolateral membrane of
the polarized cells in the intestine, kidney, liver, and placenta. NaDC1 is localized to the apical membrane of the intestinal and renal
tubular cells. In contrast, NaDC3 is localized to the basolateral membrane of the renal tubular cells, sinusoidal membrane of the hepatocytes, and the brush border membrane of the placental
syncytiotrophoblast (26). The physiological function of NaDC1 in the
intestine and kidney is to facilitate the absorption of exogenous
dicarboxylates in the intestine and the reabsorption of endogenous
dicarboxylates in the kidney. In the liver and placenta, NaDC3 may play
a role in the cellular entry of circulating dicarboxylates for
subsequent metabolic utilization. Because these dicarboxylates are
present in the circulation only in micromolar concentrations, the high affinity transporter NaDC3 has obvious advantages over the low affinity
transporter NaDC1 to perform this function. In C. elegans, the low affinity transporter NaDC1 as well as the high affinity transporter NaDC2 are expressed predominantly in the intestinal tract.
The C. elegans intestinal tract is a tubular structure made
up of a single layer of 20 donut-shaped cells (28). Unlike in mammals,
the intestinal tract in C. elegans performs a variety of
functions in addition to the digestion and absorption of dietary nutrients. It is a primary site of synthesis and storage of fat as the
energy source, a function similar to that of liver and adipose tissue
in mammals. The cells of the intestinal tract in C. elegans
are polarized, with numerous microvilli on the luminal surface
analogous to the apical membrane of the enterocytes in mammals. The
basolateral membrane of the intestinal cells is in contact with the
pseudocoelomic space that is filled with fluid that supplies nutrients
to the rest of the cells in the body. In this respect, there is a lot
of similarity between the intestinal tract in C. elegans and
the liver and adipose tissue in mammals. We have provided evidence in
this paper in support of coexpression of NaDC1 and NaDC2 in the cells
of the intestinal tract in C. elegans. It is not known,
however, whether these two transporters are distributed differentially
in the apical versus basolateral membrane of the intestinal cells.
The physiological functions of NaDC1 and NaDC2 in C. elegans
intestinal tract are not known. We used the RNAi technique to silence
the function of these two transporters to evaluate their influence on
life span in this organism. This technique is very effective in
silencing the function of any specific protein as evidenced by the
doubling of the average life span by silencing the function of DAF-2.
Homozygous mutations in daf-2 gene lead to doubling of life
span in C. elegans (23, 24). Since RNAi-mediated targeting
of daf-2 also doubles the life span, we conclude that this
technique is very effective in silencing the function of any targeted
gene. RNAi-mediated interference of NaDC1 function does not have any
noticeable effect on the average life span as well as on the maximal
life span, whereas targeting NaDC2 by this approach results in a
significant increase in the average and maximal life span. We speculate
that NaDC2 is localized to the basolateral membrane of the intestinal
cells where it functions in the cellular entry of endogenous
dicarboxylates for subsequent metabolic utilization and energy
production. Interference with this function leads to a metabolic state
analogous to that of caloric restriction, thus resulting in life span
extension. It has been well established in C. elegans that
caloric restriction (29) or suppression of metabolic energy production
within the mitochondria (30) is associated with a significant increase in life span.
It is interesting to note that Indy gene is expressed in
Drosophila in tissues such as the fat body, midgut, and
oenocytes (12, 15). The fat body in this organism is involved in the metabolism and storage of major energy sources (fat, glycogen, and
protein). The metabolic functions of this organ are similar to those of
liver in mammals. The same is true with the intestinal tract in
C. elegans where NaDC2 expression is seen. However, the transport characteristics of ceNaDC2 are very different from those of
Drosophila INDY even though disruption of NaDC2 function
enhances life span in C. elegans as disruption of INDY does
in Drosophila. In addition to NaDC1 and NaDC2 reported in
this paper, a recent search of the C. elegans data base has
revealed that there are three other genes coding for putative
transporters with structural similarity to Drosophila INDY.
Cloning and functional characterization of these putative transporters
will be required to establish the molecular identity of the gene that
is the C. elegans functional counterpart of
Drosophila INDY. The present studies have clearly shown that
NaDC2 is involved in the regulation of life span in C. elegans, but it is likely that additional transporters with NaDC2-like transport function may exist in this organism and function in the regulation of life span.