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Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
The life-extending gene Indy encodes an exchanger for Krebs-cycle
Felix Knauf1,2, Nilufar Mohebbi1, Carsten Teichert1, Diana Herold1, Blanka Rogina3, Stephen Helfand3, Maik Gollasch1, Friedrich C. Luft1 and Peter S. Aronson2* 1Franz Volhard Clinic at the Max Delbruck Center, HELIOS Kliniken – Berlin, Medical Faculty of the Charité, Humboldt University, D-13125 Berlin, Germany 2Departments of Internal Medicine and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520-8029, USA 3Department of Genetics and Developmental Biology, School of Medicine, University of Connecticut Health Center, Farmington CT 06030, USA *To whom all correspondence should be addressed: Peter S. Aronson, M.D. Department of Medicine, Section of Nephrology Yale University School of Medicine 1 Gilbert Street, TAC S-255 Box 208029 New Haven, CT 06520-8029 Telephone: 203-785-4902 Fax: 203-785-3756 Email: [email protected] Running title: INDY-mediated anion exchange Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
SYNOPSIS
A longevity gene called Indy (for "I'm not dead yet"), with homology to mammalian genes encoding Na-dicarboxylate cotransporters, was identified in Drosophila melanogaster. Functional studies in Xenopus oocytes showed that INDY mediates the flux of dicarboxylates and citrate across the plasma membrane but the specific transport mechanism mediated by INDY was not identified. To test if INDY functions as an anion exchanger, we examined whether substrate efflux is stimulated by transportable substrates added to the external medium. Efflux of [14C]-citrate from INDY-expressing oocytes was greatly accelerated by the addition of succinate to the external medium, indicating citrate-succinate exchange. The succinate-stimulated [14C]-citrate efflux was sensitive to inhibition by 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS), as previously demonstrated for INDY-mediated succinate uptake. INDY-mediated efflux of [14C]-citrate was also stimulated by external citrate and oxaloacetate, indicating citrate-citrate and citrate-oxaloacetate exchange. Similarly, efflux of [14C]-succinate from INDY-expressing oocytes was stimulated by external citrate, alpha-ketoglutarate and fumarate, indicating succinate-citrate, succinate-alpha-ketoglutarate, and succinate-fumarate exchange, respectively. Conversely, when INDY-expressing Xenopus oocytes were loaded with succinate and citrate, [14C]-succinate uptake was markedly stimulated, confirming succinate-succinate and succinate-citrate exchange. Exchange of internal anion for external citrate was markedly pHo-dependent, consistent with the concept that citrate is co-transported with a proton. Anion exchange was sodium-independent. We conclude that INDY functions as an exchanger of dicarboxylic and tricarboxylic Krebs cycle intermediates. The effect of decreasing INDY activity, as in the long-lived Indy mutants, may be to alter energy metabolism in a manner that favors life span extension. Key words: citrate, succinate, dicarboxylate, anion exchange, aging Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
INTRODUCTION

Aging is a complex biological process and involves both genetic and environmental factors. Single gene mutations affecting longevity can be a useful tool for gaining insight into the mechanisms underlying aging [1-3]. Mutations in the Indy gene (for I' m not dead yet) result in an 80–100% increase in the average life span of both adult male and female fruit flies (Drosophila melanogaster) without sacrificing fertility or physical activity [1]. The dramatically extended lifespan in Indy mutant flies is associated with a great reduction in the levels of Indy mRNA and INDY protein that are likely to result in decreased Indy activity. The life-extending Indy mutation is associated with a decrease in the slope of the mortality curve, implying that the Indy mutation causes a slowing in the demographic rate of aging and may directly affect the normal aging process [4]. Studies in Caenorhabditis elegans revealed that the disruption of transporters with similar transport properties to INDY also extends the animal's lifespan [5], demonstrating that the longevity effect of decreasing INDY activity may be conserved across distant evolutionary species [1]. Sequence analysis of Indy demonstrated closest homology to mammalian sodium- dicarboxylate cotransporters. Functional studies in Xenopus oocytes showed that INDY indeed functions as a dicarboxylate and tricarboxylate transporter [6, 7]. Kinetic measurements indicated that INDY is a relatively high-affinity transporter as the Km for succinate was approximately 40 µM [6, 7], a value similar to that of high affinity mammalian sodium dicarboxylate cotransporters [8]. In addition, immunocytochemical studies demonstrated that INDY is most prominently expressed on the basolateral membrane of cells in the midgut of Drosophila melanogaster and the plasma membrane of fat body and oenocytes [6], confirming that INDY is a plasma membrane transporter in native tissues. The polarized expression of INDY on the basolateral membrane of the midgut is similar to the membrane site of expression of high affinity mammalian sodium-dicarboxylate cotransporters [8, 9]. Despite its many similarities to mammalian sodium-dicarboxylate cotransporters, several functional properties of INDY were novel for this gene family. Importantly, organic anion transport mediated by INDY is Na-independent [6, 7], in marked contrast to sodium-dicarboxylate cotransporters. In addition, electrophysiological studies detected no electrical current associated with INDY-mediated succinate transport [7], suggesting an electroneutral transport mechanism in contrast to the electrogenic transport mediated by sodium-dicarboxylate cotransporters. Finally, INDY-mediated transport is highly sensitive to inhibition by 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) [6], which does not strongly inhibit mammalian sodium-dicarboxylate cotransporters. However, the specific transport mechanism mediated by INDY was not identified. The purpose of the present study was to test if INDY functions as a Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
DIDS-sensitive anion exchanger, and to test the effect of pH and sodium on a possible exchange process. Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
MATERIAL AND METHODS

INDY expression in Xenopus oocytes
Mature female Xenopus laevis frogs (Kaehler, Hamburg, Germany) were subjected to partial
ovarectomy under tricane (3-aminobenzoic acid ethyl ester) anesthesia (0.2% for 5-10 min) as
previously described [6, 10]. In brief, a small incision was made in the abdomen and a lobe of
ovary was removed. Subsequently, the oocytes were prewashed for 20 min in Ca-free hypotonic
medium (85 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES titrated with Tris base to pH
7.5) to remove blood and damaged tissue. Oocytes then were defolliculated by treatment with 2
mg/ml collagenase (Sigma type I) in Ca-free hypotonic solution for 45-90 min with gentle
agitation at room temperature. After this treatment, oocytes were washed three times in Ca-free
hypotonic media, and then washed three times in isotonic Ca2+-containing frog Ringer solution
(96 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5 mM HEPES, pH 7.5).

Indy cDNA was subcloned into the pGH19 expression plasmid as previously described [6]. Plasmid DNA was linearized by XhoI digestion, and cRNA was transcribed by using T7
RNA polymerase (mMESSAGE mMACHINE, Ambion, Austin, TX). Precipitated cRNA was
dissolved in sterile H2O, and yield and quality were assessed by spectroscopy and agarose gel
electrophoresis. On the day of their isolation, oocytes were injected with 50 nl of sterile H2O or
50 nl of a cRNA solution containing 25 ng of Indy cRNA by use of a Drummond microinjector.
The injected oocytes were incubated in Ca2+-containing frog Ringer solution at 18°C for
approximately 48 h to allow for expression of INDY protein.
Measurements of radiolabeled solute fluxes
For efflux experiments, the respective isotope was microinjected into the oocytes as originally
described by Stewart et al. [11]. In brief, individual oocytes were injected with 50 nl of 3.4 mM
[14C]-citrate or 3.5 mM [14C]-succinate (Amersham Biosciences, UK; MP Biomedicals, USA).
10 – 15 oocytes were lysed after the injection was completed and counted prior to the experiment
(representing 4,000–7,000 total counts per minute (cpm) per oocyte). After approximately 5 min
recovery and 2 washes in NaCl-buffer (100 mM NaCl / 5 mM Hepes / pH 7.5), the efflux assay
was initiated by transfer of individual oocytes to a 48 well plate (Beckman, Dickenson), each
well containing 1 ml of efflux solution. After 3 min, the efflux solution was removed for
scintillation counting. Radioisotope content of the efflux solution was measured by scintillation
spectroscopy after addition of 5 ml scintillation fluid (Opti-Fluor, Packard).
For uptake experiments, oocytes were microinjected with 50 nl of either water or a 1 M solution of sodium lactate, sodium succinate or sodium citrate. After approximately 5 min recovery, oocytes were washed twice in NaCl-buffer and then resuspended in the same medium Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
containing [14C]-succinate (18 µM). After a 3-min incubation, external isotope was removed by
washing the oocytes three times with ice-cold buffer. Radioisotope content of each individual
oocyte was measured by scintillation spectroscopy after solubilization in 0.5 ml of 10% (vol/vol)
SDS and addition of 5 ml scintillation fluid.

Statistical analysis
Results shown in the bar graphs represent means ± S.E.M. (standard error of the mean) for 8-16
oocytes. The results were analyzed using non-paired Student's t-test. P values < 0.05 were
considered statistically significant.
Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
RESULTS

Effects of Krebs cycle intermediates on [14C]-citrate efflux
As an initial approach to examine whether INDY can mediate the exchange of dicarboxylic and
tricarboxylic Krebs cycle intermediates, we microinjected [14C]-citrate into INDY-expressing
oocytes, and tested whether organic anions previously found to be transported by INDY [6, 7]
can stimulate [14C]-citrate efflux. As illustrated in Fig. 1, the presence of 1 mM succinate added
to the external medium greatly stimulated the efflux of [14C]-citrate compared to the control in
which 1 mM lactate was added to the external solution. Lactate has been previously shown to
have no detectable affinity for INDY [6]. The INDY-mediated citrate efflux was almost
completely abolished by 0.1 mM DIDS, which has previously been shown to inhibit INDY-
mediated dicarboxylate and tricarboxylate uptake [6]. Importantly, external succinate failed to
stimulate [14C]-citrate efflux from water-injected oocytes not expressing INDY, confirming that
the DIDS-sensitive citrate-succinate exchange is mediated by INDY and not by an endogenous
transporter in Xenopus oocytes.
Previous studies have shown that INDY-mediated succinate and citrate uptake is not cation-dependent [6, 7]. In order to evaluate whether INDY-mediated citrate-succinate exchange is also Na-independent, we measured succinate stimulated [14C]-citrate efflux in the presence and absence of Na in the external buffer. The stimulation of efflux of [14C]-citrate by external succinate was not altered by the replacement of sodium with potassium (not shown), confirming that INDY mediated citrate-succinate exchange is Na-independent. Having demonstrated that INDY functions as an anion exchanger, we next examined whether additional INDY substrates added to the external medium can also stimulate [14C]-citrate efflux. Similar to succinate, the presence of 1 mM unlabeled citrate in the external medium greatly stimulated DIDS-sensitive [14C]-citrate efflux, consistent with INDY-mediated citrate-citrate exchange. Similarly, the addition of oxaloacetate to the external medium also greatly stimulated the DIDS-sensitive efflux of [14C]-citrate, indicating INDY-mediated citrate-oxaloacetate exchange. Effects of Krebs cycle intermediates on [14C]-succinate efflux
To evaluate the reversibility and anion specificity of INDY-mediated anion exchange in more
detail, we tested the ability of INDY to mediate efflux of [14C]-succinate in exchange for external
dicarboxylates and tricarboxylates. As illustrated in Fig. 2, the presence of 1 mM external citrate
greatly stimulated the DIDS-sensitive efflux of [14C]-succinate from INDY-expressing oocytes,
consistent with succinate-citrate exchange. External citrate failed to stimulate [14C]-succinate
efflux from water-injected oocytes not expressing INDY, confirming that the DIDS-sensitive
Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
citrate-succinate exchange is mediated by INDY and not by an endogenous transporter in Xenopus oocytes. Taken together, the results in Fig. 1 and Fig. 2 indicate that INDY-mediated tricarboxylate-dicarboxylate exchange is reversible, as exchange of internal citrate for external succinate, and internal succinate for external citrate, were both demonstrated. We next tested the ability of INDY to exchange dicarboxylates of unequal chain lengths. As also shown in Fig. 2, the presence of 1 mM external alpha-ketoglutarate strongly stimulated the DIDS-sensitive efflux of [14C]-succinate, indicating that INDY can mediate succinate-alpha-ketoglutarate exchange. These findings confirmed that INDY is capable of exchanging dicarboxylates of unequal chain lengths across the plasma membrane. In addition, we tested the ability of INDY to mediate exchange of dicarboxylates with different redox states. The addition of 1 mM fumarate to the external solution greatly stimulated
DIDS-sensitive [14C]-succinate efflux, consistent with succinate-fumarate exchange (Fig. 2).
These findings indicate that INDY can exchange dicarboxylates of different redox states.
Effects of Krebs cycle intermediates on [14C]-succinate influx
To provide additional confirmation for the presence of anion exchange, we investigated whether
pre-loading of INDY-expressing oocytes with citrate and succinate facilitates the influx of [14C]-
succinate. As illustrated in Fig. 3, injection of lactate into INDY-expressing oocytes failed to
stimulate [14C]-succinate uptake compared to INDY-expressing oocytes injected with an equal
volume of water. Pre-loading of INDY-expressing oocytes with succinate and citrate greatly
stimulated [14C]-succinate uptake, and this stimulation by internal anions was completely
abolished by DIDS. These findings confirm the presence of DIDS-sensitive succinate-succinate
and succinate-citrate exchange.
Effects of pH on succinate- and citrate-mediated [14C]-citrate efflux
We had previously observed that INDY-mediated citrate uptake is stimulated at pH 6.0 but
INDY-mediated succinate uptake is pH-independent [6]. It was therefore of interest to test the
effect of pH on the ability of external citrate and succinate to stimulate anion efflux by anion
exchange. Shown in Fig. 4, the effect of external citrate to stimulate [14C]-citrate efflux from
INDY-expressing oocytes was markedly enhanced at pH 6.0 when compared to pH 7.5. In
contrast, the effect of external succinate to stimulate [14C]-citrate efflux was pH-independent. At
pH 7.5, citrate (pK values 3.1, 4.8, 6.4) is predominantly in the tricarboxylate form whereas
succinate (pK values 4.2, 5.6) is predominantly in the dicarboxylate form. Thus, these results
indicate either that external citrate is preferentially exchanged in the dicarboxylate form and/or
that the tricarboxylate form is exchanged together with a proton.
Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409

DISCUSSION

We have demonstrated that the transporter encoded by the life-extending gene Indy is a
disulfonic stilbene-sensitive, Na-independent, anion exchanger capable of exchanging pairs of
dicarboxylates and citrate across the plasma membrane. Our findings on the effects of pH
suggest that citrate is preferentially exchanged in the dicarboxylate form and/or that the
tricarboxylate form is transported together with a proton, whereas transport of dicarboxylates is
H+-independent. The function of INDY as an obligate anion exchanger can explain the
previously observed electroneutrality of INDY-mediated anion transport [7]. Dicarboxylate-
dicarboxylate exchange, citrate-citrate exchange, and exchange of a dicarboxylate for citrate with
a proton are all predicted to be electroneutral processes. The function of INDY as an obligate
anion exchanger is in contrast to that of the mammalian sodium-dicarboxylate cotransporters,
which utilize the Na gradient to drive net uphill anion transport across the plasma membrane [12-
14]. Thus, INDY defines a different class of transporter, exchanging Krebs-cycle intermediates
across the plasma membrane independently of sodium.
Interestingly, the function of INDY as an electroneutral anion exchanger of dicarboxylates for citrate plus a proton is very similar to that of the citrate transport protein of the mitochondrial inner membrane [15, 16]. Although INDY shows no significant sequence homology to the mitochondrial citrate transport protein [17], INDY is capable of mediating exchange reactions described for the citrate transport protein, including citrate-succinate exchange (Fig. 1, Fig. 2 and Fig. 3), citrate-oxaloacetate exchange (Fig. 1), or citrate–citrate exchange (Fig. 1). In addition to tricarboxylate-dicarboxylate exchange, INDY is also capable of exchanging dicarboxylates of unequal chain lengths such as succinate for alpha-ketoglutarate (Fig. 2), and dicarboxylates of different redox states such as succinate for fumarate (Fig. 2). Fatty acid synthesis predominantly occurs in the cytoplasm of the cell and requires acetyl-CoA as the main source. The mitochondrial citrate transport protein plays a major role in this process [17, 18]. INDY, which is primarily expressed on the plasma membrane of cells of the midgut and fat body of the fruit fly [6], might have an equally important role in importing citrate from hemolymph into the cells. Citrate can then be cleaved by citrate lyase to acetyl-CoA and used for fatty acid synthesis [18]. The cytosolic pool of citrate is in common for both mitochondrial and plasma membrane transport systems. Thus, INDY might fulfill a physiological function similar to the mitochondrial citrate transport protein in overall cellular metabolism by regulating the amount of citrate in the cytosol for lipid synthesis. A potential role for INDY in lipid metabolism is supported by the recent finding in C. elegans that decreased Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
expression of an INDY homologue, the Na-citrate cotransporter ceNaC-2, results in a reduction in fat content and an increase in life span [19]. Given that INDY is a reversible anion exchanger, the net transfer of citrate and caloric or redox equivalents across the plasma membrane mediated by INDY will depend on the relative concentrations of transportable substrates both within the cell and in the extracellular fluid. In the absence of such information, we cannot predict with certainty the effect that a defect in INDY activity would have on cellular metabolism. Nevertheless, the ability of INDY to mediate net transfer of citrate and caloric or redox equivalents across the plasma membrane is consistent with the universal role of energy balance in aging as documented in multiple model systems (e.g. worms, flies, rodents) [20]. This concept is supported by the aforementioned observation that knockdown of expression of the homologous citrate transporter ceNaC-2 increases the life span of worms [5, 19]. Thus, it is likely that a reduction in INDY activity in mutant Indy long-lived flies alters energy metabolism in a manner similarly conducive to life span extension. Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
ACKNOWLEDGEMENTS
Support was provided by a stipend from the Max-Delbruck Center for Molecular Medicine and
the Studienstiftung des Deutschen Volkes to F.K, a grant-in-aid to F.C.L and M.G. from the
Deutsche Forschungsgemeinschaft, a Helmholtz Gesellschaft Fellowship to M.G., NIH grants
AG-14532 to S.L.H., DK-17433 and DK-33793 to P.S.A., AG-23088 to B. R., an Investigator
Award from The Patrick and Catherine Weldon Donaghue Medical Foundation to S.L.H., and an
Ellison Medical Foundation Senior Investigator Award to S.L.H.
Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
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FIGURE LEGENDS
Figure 1. Effect of external substrates on [14C]-citrate efflux from INDY-expressing
Xenopus oocytes.
INDY-expressing oocytes and water-injected oocytes not expressing INDY were microinjected
with [14C]-citrate and then washed and transferred to different external solutions: NaCl buffer
with 1 mM of the indicated substrate, or NaCl buffer with 1 mM of the indicated substrate and
100 µM DIDS. The appearance of radioactivity in the external solution during a 3-min
incubation was determined by scintillation spectroscopy. Data are means ± SEM. * indicates p <
0.05. N.S. indicates not statistically significant.
Figure 2. Effect of external substrates on [14C]-succinate efflux from INDY-expressing
Xenopus oocytes.
INDY-expressing oocytes and water-injected oocytes not expressing INDY were microinjected
with [14C]-succinate and then washed and transferred to three different external solutions: NaCl
buffer with 1 mM of the indicated substrate, or NaCl buffer with 1 mM of the indicated substrate
and 100 µM DIDS. The appearance of radioactivity in the external solution during a 3-min
incubation was determined by scintillation spectroscopy. Data are means ± SEM. * indicates p <
0.05. N.S. indicates not statistically significant.
Figure 3. Effect of internal substrates on [14C]-succinate uptake into INDY-expressing
Xenopus oocytes.
INDY-expressing oocytes were microinjected with 50 nl of H20 or a 1 M solution of the
indicated substrate. Oocytes were then washed and transferred to NaCl-buffer containing [14C]-
succinate ± 100 µM DIDS. After 3-min incubation, external isotope was removed by washing
the oocytes with ice-cold buffer and radioisotope content was measured by scintillation
spectroscopy. Data are means ± SEM. * indicates p < 0.05. N.S. indicates not statistically
significant.
Figure 4. Effect of external pH on citrate versus succinate stimulated [14C]-citrate efflux
from INDY-expressing Xenopus oocytes.
INDY-expressing oocytes were microinjected with [14C]-citrate and then washed and transferred
to different external solutions: NaCl buffer with 1 mM succinate versus citrate at pH 6.0, pH 7.5
and pH 9.0. The appearance of radioactivity in the external solution during a 3-min incubation
was determined by scintillation spectroscopy. Data are means ± SEM. N.S. indicates not
statistically significant.
Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
Copyright 2006 Biochemical Society
Biochemical Journal Immediate Publication. Published on 12 Apr 2006 as manuscript BJ20060409
Copyright 2006 Biochemical Society

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