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J. Biol. Chem., Vol. 282, Issue 16, 11996-12009, April 20, 2007

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Zebrafish Slc5a12 Encodes an Electroneutral Sodium Monocarboxylate Transporter (SMCTn)

A COMPARISON WITH THE ELECTROGENIC SMCT (SMCTe/Slc5a8)^*

Consuelo Plata¹, Caroline R. Sussman^¶, Aleksandra Sini^¶1, Jennifer O. Liang^||, David B. Mount^**2, Zara M. Josephs, Min-Hwang Chang, and Michael F. Romero^¶3

From the Departments of Physiology and Biophysics and ^||Biology, Case Western Reserve University, Cleveland, Ohio 44106, Departamento de Nefrología y Metabolismo Mineral, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Tlalpan 14000, México City, México, ^¶Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN 55905, and ^**Renal Division, Brigham and Women's Hospital and Division of General Internal Medicine, Veterans Affairs Boston Healthcare System, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, October 2, 2006 , and in revised form, January 18, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We have identified and characterized two different sodium-coupled monocarboxylate cotransporters (SMCT) from zebrafish (Danio rerio), electrogenic (zSMCTe) and electroneutral (zSMCTn). zSMCTn is the 12th member of the zebrafish Slc5 gene family (zSlc5a12). Both zSMCT sequences have 50% homology to human SLC5A8 (hSMCT). Transport function and kinetics were measured in Xenopus oocytes injected with zSMCT cRNAs by measurement of intracellular Na⁺ concentration ([Na⁺]_i) and membrane potential. Both zSMCTs oocytes increased [Na⁺]_i with addition of monocarboxylates (MC) such as lactate, pyruvate, nicotinate, and butyrate. By using two electrode voltage clamp experiments, we measured currents elicited from zSMCTe after MC addition. MC-elicited currents from zSMCTe were similar to hSMCT currents. In contrast, we found no significant MC-elicited current in either zSMCTn or control oocytes. Kinetic data show that zSMCTe has a higher affinity for lactate, nicotinate, and pyruvate (K_m^L-lactate = 0.17 ± 0.02 mM, K_m^nicotinate = 0.54 ± 0.12 mM at -150 mV) than zSMCTn (K_m^L-lactate = 1.81 ± 0.19 mM, K_m^nicotinate = 23.68 ± 4.88 mM). In situ hybridization showed that 1-, 3-, and 5-day-old zebrafish embryos abundantly express both zSMCTs in the brain, eyes, intestine, and kidney. Within the kidney, zSMCTn mRNA is expressed in pronephric tubules, whereas zSMCTe mRNA is more distal in pronephric ducts. zSMCTn is expressed in exocrine pancreas, but zSMCTe is not. Roles for Na⁺-coupled monocarboxylate cotransporters have not been described for the brain or eye. In summary, zSMCTe is the zebrafish SLC5A8 ortholog, and zSMCTn is a novel, electroneutral SMCT (zSlc5a12). Slc5a12 in higher vertebrates is likely responsible for the electroneutral Na⁺/lactate cotransport reported in mammalian and amphibian kidneys.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The liver and the kidneys are the major sites of lactate uptake and metabolism. Normally the blood lactate concentration is 1.2 mM; however, lactate levels can increase to 10 mM during maximal exercise. Lactate is reabsorbed in kidney such that only trace amounts of lactate are normally excreted in the urine. Lactate in the glomerular ultrafiltrate is almost completely reabsorbed by the proximal nephron (1).

The movement of lactate and other monocarboxylates across plasma membranes is thought to occur by proton-linked monocarboxylate transporters (MCT)⁴ (2, 3). The MCT gene family (Slc16) now includes 14 members, of which only the first 4 members (MCT1–MCT4) have been demonstrated experimentally to mediate the proton-linked transport of metabolically important monocarboxylates, such as lactate, pyruvate, and short chain fatty acids. MCTs are expressed in a number of tissues, including kidney (2, 3). However, a significant body of data indicates an important role for luminal Na⁺/monocarboxylate cotransport in the reabsorption of filtered lactate by the proximal tubule. Some studies have concluded that luminal Na⁺/lactate transport is electroneutral (4, 5), whereas other reports have shown that the process is electrogenic (6–8). In the kidney, absorption of pyrazinoate, an antituberculosis drug, and nicotinate (also known as niacin), used in the treatment of dyslipidemias (9–11), occurs via an electroneutral Na⁺/monocarboxylate transport system (12) suggesting multiple transporters. Thus, transport of these compounds and structurally similar compounds by SMCT is likely of immediate clinical importance.

SLC5A8, a member of the Na⁺/glucose cotransporter family, was recently reported to function as an electrogenic sodium monocarboxylate transporter (13, 14). The Slc5 gene family encodes versatile cotransporters of organic solutes, ions, and potentially water (15). In humans, 11 SLC5 genes have been identified (15). SLC5 members function as Na⁺ and solute (e.g. glucose, myoinositol, iodide and multivitamins) cotransporters (15). Although the functions of some of the family members are well understood, e.g. Slc5a1 (SGLT1; the Na⁺/glucose cotransporter) and Slc5a5 (NIS; the Na⁺/I^- cotransporter), others family members (SLC5A9-A12) have not been well characterized (15, 16).

SLC5A8 was originally cloned by Rodriguez et al. (17) in an attempt to identify iodide transporters that might play a functional role in the physiology of the thyroid gland. This gene codes for a protein with 46% identity in amino acid sequence with NIS (Slc5a5) (18). The SLC5A8 protein is expressed in the apical membrane of thyroid follicular cells and was hypothesized to mediate passive transport of iodide independent of Na⁺; consequently, Slc5a8 was originally named the "apical iodide transporter" (17).

Recently, the SLC5A8 gene was shown to play an important role in controlling the development of colon cancers (19) and classical papillary thyroid carcinomas (PTC-cf) (20). Silencing of SLC5A8 is a common and early event in both tumors. Promoter methylation that inactivates SLC5A8 is found in 60% of colon cancer cell lines and primary colon cancers and >80% adenomas (19). In the case of PTC-cf, SLC5A8 silencing by methylation was found in 90% of thyroid carcinomas (follicular, the most common type) and occurred in about 20% of other papillary thyroid carcinomas (20). SLC5A8 is thus also hypothesized to be a tumor suppressor gene. We reasoned that such important functions of SLC5A8 should be evolutionarily preserved and could be crucial in development and differentiation, particularly of epithelial tissues. In fact, Costa and co-workers (21) have found that Slc5a8 (Vito) is first expressed at the blastopore lip (the beginning of neurulation and gastrulation) in Xenopus tadpoles.

Human SLC5A8 and mouse Slc5a8 protein functions have been studied previously in Xenopus laevis oocytes. As a transporter, SLC5A8 is an electrogenic Na⁺/monocarboxylate cotransporter (SMCT) moving substrates such as short chain fatty acids (propionate, butyrate, and valerate) as well as monocarboxylates such as lactate, pyruvate, nicotinate, and pyrazinoate (13, 14, 22). Yet, these substrates are apparently moved with varied affinity and coupling to Na⁺ (13, 14, 22).

Slc5a8 was also cloned from mouse kidney (23). Not surprisingly, mouse Slc5a8 expressed in Xenopus oocytes is electrogenic, transporting the same substrates as human SMCT (13, 14). Thus, Slc5a8 is the first transporter known to be expressed in mammalian colon and kidney that has the ability to mediate the electrogenic absorption of lactate and other monocarboxylates with Na⁺ (13, 23, 24). The K_m and V_max values for MC substrates of SLC5A8 indicate that it is a high affinity and low capacity transporter that could easily be saturated by MCs in the renal ultrafiltrate or in the colonic lumen. Thus, an electroneutral Na⁺/MC cotransporter, particularly with high transport capacity, would allow MC absorption without excess loss in the urine or stool. Yet the molecular identity of such electroneutral Na⁺/lactate transport proteins in kidney remains unknown.

In this study we identified, localized, and functionally characterized two zebrafish SMCTs. One is the electrogenic SMCT (Slc5a8), and the other is a novel electroneutral form of the Na⁺/monocarboxylate cotransporter (SMCTn) with high transport capacity. SMCTn is the zebrafish ortholog of Slc5a12 recently reported as a second electrogenic Na⁺/monocarboxylate cotransporter (32). Our data demonstrate that both SMCTn and the electrogenic SMCT (SMCTe/Slc5A8) are present in kidney and brain of the teleost Danio rerio (zebrafish) and have different kinetic properties. Both proteins increase intracellular Na⁺ in response to a variety of MCs as follows: short chain fatty acids, lactate, pyruvate, nicotinate, acetoacetate, and 3--hydroxybutyrate. Slc5a8 mRNA is also found in the gut consistent with its original description in human.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
cDNA Cloning of Zebrafish SMCT, zSlc5a8 and zSlc5a12—A BLAST search of the EST data base revealed two D. reiro cDNAs that were homologous to human SLC5A8, yet clearly distinct from one another. The cDNAs corresponding these ESTs (IMAGE clones zSMCTe (zSlc5a8, 7212813) and zSMCTn (Slc5a12, 6793401)) were obtained from the IMAGE consortium (Research Genetics, Genome Systems) and sequenced (GenBank^TM accession numbers AY727859 [GenBank] for zSMCTe and AY727860 [GenBank] for zSMCTn). The complete EST inserts were sequenced (W. M. Keck Facility, New Haven, CT) and then subcloned in pGEMHE Xenopus oocyte expression vector between EcoRI (5') and XbaI (3').

Oocyte Isolation and Injection—Oocytes were removed from female X. laevis (Xenopus Express, Beverly Hills, FL) as described previously (25). Excised lobes of oocytes were placed into a Ca²⁺-free buffered saline solution (200 mosM) and defolliculated by collagenase digestion as described previously (26). Capped zSlc5a8 and zSlc5a12 cRNAs were synthesized using a linearized cDNA template and the T7 mMessage mMachine (Ambion, Austin, TX). Oocytes were injected with 50 nl of 0.5 ng/nl (25 ng/oocyte) cRNA of zSMCTe, zSMCTn, hSMCT, or water. The oocytes were maintained at 18 °C in filtered ND96 medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES-Tris (pH 7.5)) (13). Oocytes were studied 3–10 days after injection. All experiments were performed in accordance with the regulations of the Institutional Animal Care and Use Committee of Case Western Reserve University.

Electrophysiology—All monocarboxylate solutions (1 mM lactate, pyruvate, propionate, and butyrate) were prepared in ND96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES (pH 7.5)) as described previously (13). Unless otherwise stated, MCs were all used at 1 mM. To examine the kinetics of lactate and nicotinate transport of zSMCTn, a low chloride ND96 was used to maintain the same tonicity for all solutions (200 mosmol). For lactate curves, we used (in mM) 66 NaCl base solution with 30 to 0 sodium gluconate and 0 to 30 sodium lactate. For nicotinate curves, we used 56 NaCl base with 50 to 0 sodium gluconate and 0–50 sodium nicotinate. All solutions also contained 2 KCl, 1.8 CaCl₂, 1 MgCl₂, 5 HEPES and were pH 7.5.

Ion-selective Microelectrode—Ion-selective microelectrodes were used to monitor intracellular Na⁺ concentration ([Na⁺]_i) of zSMCTs and water-injected oocytes as described previously (25–27). Intracellular [Na⁺] was measured as the difference between the Na⁺ electrode and a KCl voltage electrode impaled into the oocyte; and membrane potential (V_m) was the potential difference between the KCl microelectrode and an extracellular calomel (26, 28). Na⁺ microelectrodes were calibrated using 10 mM NaCl and 100 mM NaCl solutions followed by point calibration in ND96 (96 mM Na⁺) as performed previously (26, 28). Intracellular Na⁺ microelectrodes had slopes of at least -50 mV/p(Na⁺).

Two Electrode Voltage Clamp—Oocyte membrane currents were recorded using an OC-720C voltage clamp (Warner Instruments, Hamden, CT), filtered at 2–5 kHz, digitized at 10 kHz, and recorded with the Pulse software, and data were analyzed using the PulseFit program (HEKA, Germany) as described previously (25, 29). For periods when I-V protocols were not being run, oocytes were clamped at a holding potential (V_h) of -50 mV; and the current was constantly monitored and recorded at 1 Hz. I-V protocols consisted of 400-ms steps from V_h to -150 mV and +50 mV in 20-mV steps as described previously (13).The I-V protocols were run in the absence and in the presence of a particular substrate. The substrate-specific current is determined by subtraction of the pre-substrate current from the substrate current. All of the experiments were performed at room temperature. Test substrates bathed oocytes for 1–3 min. For kinetic analysis, oocytes were exposed to only one monocarboxylate at one concentration and then discarded.

Calculations—Oocytes were perfused with ND96 for 5 min at which time "initial [Na⁺]_i" is measured. The solution was switched to different monocarboxylates for 8–10 min (i.e. [Na⁺]_i and V_m or I plateau), and the "final [Na⁺]_i" is measured. Oocytes came from at least four separate donor animals.

²²Na⁺ Uptakes—Functions of zSMCTe and zSMCTn were assessed by measuring ²²Na⁺ uptake in groups of 15–20 oocytes 4 days after water or cRNA injection. ²²Na⁺ uptake was performed as described previously (30). Briefly, ²²Na⁺ uptake was measured with the following protocol: a 30-min incubation period ND96 with 1 mM ouabain, 100 µM amiloride, 100 µM bumetanide, followed by a 60-min uptake period in ND96. To analyze the pyruvate transport kinetics, we varied pyruvate concentration from 0 to 5 mM in ND96 for zSMCTe and from 0 to 20 mM for zSMCTn. These uptake solutions also contained 1 µCi/ml of ²²Na⁺ (PerkinElmer Life Sciences) and the same inhibitors used during the incubation period. Uptakes were performed at 30 °C. At the end of the uptake period, oocytes were washed five times in ice-cold ND96 solution without isotope to remove extracellular tracer. Next, individual oocytes were dissolved in 10% SDS, and tracer activity was determined by scintillation counting. RNA-injected oocytes were compared with water controls subjected to identical conditions, using oocytes from the same donor. Kinetic analysis was performed by estimating the K_m values for pyruvate of each SMCT. The K_m values were calculated from log [ion] versus V/V_max plots using GraphPad Prism (GraphPad Software, Inc., San Diego).

RNA in Situ Hybridization—Single label in situ hybridization was carried out as described (31). Reagents were obtained from Roche Applied Science. Briefly, zebrafish embryo groups were incubated at 70 °C with digoxigenin-UTP-labeled antisense RNA probe for zSMCTe or zSMCTn in hybridization solution containing 50% formamide, detected with an anti-digoxigenin antibody conjugated to alkaline phosphatase (AP), and visualized with a combination of 4-nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (blue precipitate). We alkali-hydrolyzed cRNA probes with 0.2 M Na₂CO₃ (pH 10.2) for 14 min at 60 °C to obtain 200–300-bp fragments (size verified by RNA gel and [cRNA] determined by UV spectrometry). AP buffer is 100 mM Tris-HCl (pH 9.5), 50 mM MgCl₂, 100 mM NaCl, 0.1% Tween 20. The developing solution was 225 µg/ml 4-nitro blue tetrazolium and 175 µg/ml 5-bromo-4-chloro-3-indolyl phosphate in AP buffer. Endogenous AP activity was inhibited by adding 2 mM levamisole (Sigma) to all solutions after antibody incubation. Following in situ hybridization, embryos were postfixed in 4% paraformaldehyde, dehydrated through a graded ethanol series, destained with undiluted methyl salicylate (M6752, Sigma), digitally photographed (27), and stored in mineral oil.

Fish Embryo Sections—After in situ hybridization, 5 dpf embryos were postfixed in 4% paraformaldehyde for 1 h at room temperature. Fixed embryos were incubated overnight in 30% sucrose in phosphate-buffered saline and then embedded in OCT. 15-µm cryosections were cut with a Leica cryostat and mounted on gelatin-coated slides. Embryo sections were observed using a Zeiss Axiovert 25 microscope and acquired with an AxioCam digital camera and AxioVision software (Carl Zeiss, Germany) as described previously (27).

Statistical Analysis—The results are presented as means ± S.E. The significance of the differences between groups was tested by one-way analysis of variance with multiple comparison using Bonferroni correction or by the Kruskal-Wallis one-way analysis of variance on ranks with the Dunn method for multiple comparison procedure, as needed.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Molecular Cloning of the zSMCTs—We found two cDNAs for the zebrafish (D. rerio) homologs of the human electrogenic sodium/monocarboxylate cotransporter (hSMCTe/SLC5A8). One cDNA (zSMCTe) encodes a 610-amino acid protein (GenBank^TM accession number AY727859 [GenBank] ), whereas the other cDNA (zSMCTn) encodes a 623 amino acid protein (GenBank^TM accession number AY727860 [GenBank] ). zSMCTe and zSMCTn are 46% identical. Both zSMCT sequences are roughly equally divergent from mammalian Slc5a8. zSMCTe has 55 and 56% amino acid identity, and zSMCTn has 51 and 53% amino acid identity to human and mouse Slc5a8, respectively. zSMCTn shows 64% identity to mouse Slc5a12, and zSMCTe is 48% identical to mammalian Slc5a12 (Fig. 1).

Secondary structure of zSMCT proteins was predicted using TMHMM. This analysis predicts that zSMCTe has 13 transmembrane spans, as indicated for hSMCT (19). The zSMCTe protein contains three potential N-linked glycosylation sites in extracellular loops (Asn-112, Asn-439, and Asn-485), 19 Ser, 8 Thr, and 4 Tyr-like potential phosphorylation sites but only 7 Ser (Ser-477, Ser-523, Ser-541, Ser-556, Ser-568, Ser-570, and Ser-588), 2 Thr (Thr-75 and Thr-84), and one Tyr (Tyr-80) are outside of the membrane, increasing the probability these are actual phosphorylation sites.

View larger version (12K): [in this window] [in a new window]   FIGURE 1. Homology of known SMCTs. Slc5a8 and Slc5a12 are most related to NIS (Slc5a5, not shown) in the Slc5 gene family. However, these two monocarboxylate transporters (SMCTe and SMCTn) form an independent branch of the Slc5 family. The two main branches of the monocarboxylate arm of the Slc5 gene family are outlined. The length of the horizontal lines approximates evolutionary distance. Phylogenetic tree was generated using the DNAstar program (Madison, WI). Figure illustrates that both sequences of zSMCT are 50% identical to human and mouse SMCTe (Slc5a8), but zSMCTn is 65% identical to human and mouse Slc5a12.

Functional Expression of zSMCT Proteins in Xenopus Oocytes— We analyzed the function of both zSMCT clones by simultaneous measurement of membrane potential (V_m) and intracellular Na⁺ concentration ([Na⁺]_i) with ion-selective microelectrodes in Xenopus oocytes injected with zSMCTn or zSMCTe cRNA. Using two electrode voltage clamp experiments, we also examined currents elicited by monocarboxylates for both SMCT cotransporters.

All MCs tested induced a significant increase of intracellular [Na⁺] in both zSMCT oocytes ([Na⁺] ^zSMCTe_i = 2.76 ± 0.29 mM, n = 8; [Na⁺] ^zSMCTn_i = 2.28 ± 0.33 mM, n = 9). In contrast, control oocytes displayed only minor increments of intracellular Na⁺ ([Na⁺] ^control_i = 0.31 ± 0.10 mM; n = 6). Comparison to the human transporter indicates that hSMCTe transports slightly more Na⁺ [Na⁺]^hSMCT_i = 3.47 ± 0.77 mM) than both zSMCT clones expressed in oocytes.

Fig. 2, A–D, shows individual experiments of simultaneous measurement of [Na⁺]_i (top traces) and V_m (bottom traces) in oocytes. These experiments show that addition of 1 mM MC⁵ to a zSMCTe oocyte (Fig. 2C) increases [Na⁺]_i from 2.9 to 6.5 mM ([Na⁺]_i = 3.6 mM) and in a zSMCTn oocyte (Fig. 2D) increases [Na⁺]_i from 3.8 to 5.8 mM ([Na⁺]_i = 2.0 mM). When human SMCTe is expressed, [Na⁺]_i increases a bit more with MC addition (Fig. 2B), i.e. from 5.2 to 11.9 mM ([Na⁺]_i = 6.6 mM), whereas [Na⁺]_i, of the water-injected oocyte (Fig. 2A), was only slightly altered (3.5–4.0 mM, [Na⁺]_i = 0.5 mM). Increases of [Na⁺]_i for all SMCT clones (hSMCTe, zSMCTe, and zSMCTn) occurred only in the presence of a transported MC. When the MC tested was removed, the [Na⁺]_i increase stopped. Fig. 2 also shows that the [Na⁺]_i increment elicited by butyrate is dependent on extracellular Na⁺, i.e. when extracellular Na⁺ was removed, it elicited a rapid fall of [Na⁺]_i in all SMCT oocytes but not in controls despite the continued butyrate presence (zSMCTe =-0.58 mM, zSMCTn =-1.25 mM, hSMCTe =-0.45 mM; water =-0.13 mM). These data indicate that Na⁺ and MCs move together and are capable of moving into as well as out of the oocytes.

View larger version (40K): [in this window] [in a new window]   FIGURE 2. zSMCT expression, intracellular Na+ transport. Simultaneous measurement of intracellular Na+ concentration ([Na+]i)(top panels) and membrane potential (Vm)(bottom panels) for a water-injected control oocyte (A), hSMCTe-injected oocyte (B), zSMCTe-injected oocyte (C), and zSMCTn-injected oocyte (D). Oocytes were continually superfused as indicated under "Experimental Procedures." Monocarboxylates (1 mM pyruvate (Pyr), lactate (Lac), propionate (Pro), or butyrate (Buty)) were added as indicated by the horizontal lines. Na+ removal (0Na+) was a choline replacement. Average Na+ transport responses [Na+]i = end[Na+]i - initial[Na+]i in the presence of pyruvate (left hatched bar), lactate (horizontal hatched bar), propionate (right hatched bar), butyrate (open and gray bars), and 0Na+/butyrate (black bar) are indicated in the column graphs: water-injected oocytes (E), hSMCTe-injected oocytes, n = 8(F), zSMCTe-injected oocytes, n = 9(G), and zSMCTn-injected oocytes, n = 9(H). Data were collected from at least four frogs. *, statistical significance (p 0.05) for [Na+]i increment compared with butyrate response for that clone. **, statistically significant difference (p 0.05) of Na+-free + butyrate between both groups (hSMCTe and zSMCT). Data are reported for 6–7 oocytes from at least two frogs. Individual oocyte experiments are noted with numeric labels (below time bars) indicating the oocyte number used.

View larger version (34K): [in this window] [in a new window]   FIGURE 3. zSMCTe but not zSMCTn elicited currents in presence of monocarboxylates. Typical voltage clamp experiments are shown. zSMCTe-injected oocytes (A) are exposed to 1 mM butyrate (Buty) eliciting currents not observed in zSMCTn-injected oocytes (B). IButy was measured before (open square), during (0Na+/Butyrate, open triangle), and after (open square) removal of bath Na+. C shows zSMTCe I-V relationships (holding potential =-50 mV) for 1 mM different MCs: butyrate (open shapes), propionate (closed circle), lactate (closed triangle), and pyruvate (reverse closed triangle). Voltage step protocols were executed 30–60 s before a solution change (see "Experimental Procedures"). Step protocols were run at peak currents. IMCs were calculated as I(MCs) - I(ND96) and plotted versus pulse voltage (C). Individual voltage clamp experiments with water-injected oocytes (D and F) and zSMCTn-injected oocytes (E and G) are shown to illustrate that exposure of these oocytes to high concentrations of nicotinate (Nico, 40 mM) and L-lactate (Lac, 30 mM) did not elicit a current response.

The [Na⁺]_i increase for zSMCTe oocytes (Fig. 2C) is concurrent with large depolarization (V_m: butyrate = 46 mV, propionate = 51 mV, L-lactate = 50 mV, pyruvate = 42 mV; average responses in Fig. 2G, bottom), similar to that observed with hSMCTe oocytes (Fig. 2B) (V_m: butyrate = 45 mV, propionate = 29 mV, L-lactate = 30 mV, pyruvate = 34 mV; average responses in Fig. 2F, bottom). In contrast, zSMCTn oocytes (Fig. 2D) do not show any change in V_m (V_m: butyrate = 1.0 mV, propionate = 1.0 mV, L-lactate = 2.0 mV, pyruvate = 2.0 mV; average responses in Fig. 2H, bottom) similar to water-injected control oocytes (V_m: butyrate = 0.0 mV, propionate = 1.0 mV, L-lactate = 1.0 mV, pyruvate = 1.0 mV) (Fig. 2A; average response in Fig. 2E, bottom).

The average V_m change (V_m) (Fig. 2, bottom columns) in the presence of various MCs for nine zSMCTn oocytes (Fig. 2H) was not different between the average V_m for six control oocytes (Fig. 2E). Yet both data sets are significantly different from the average V_m for eight zSMCTe oocytes (Fig. 2G) and for nine hSMCT oocytes (Fig. 2F).

Fig 2(middle columns) also shows the average [Na⁺]_i in response to 1 mM of different MCs for water controls (Fig. 2E), hSMCTe oocytes (Fig. 2F), zSMCTe oocytes (Fig. 2G), and zSMCTn oocytes (Fig. 2H). These data show that the [Na⁺]_i of zSMCTe oocytes in the presence of pyruvate is significantly different with respect to other MCs (p = 0.05; [Na⁺]_i: butyrate = 0.58 ± 0.12 mM, propionate = 0.47 ± 0.04 mM, L-lactate = 0.64 ± 0.08 mM, pyruvate = 0.96 ± 0.11 mM). However, [Na⁺]_i data for zSMCTn oocytes is significantly different regarding [Na⁺]_i between pyruvate and L-lactate (p = 0.05; [Na⁺]_i: butyrate = 0.54 ± 0.11 mM, propionate = 0.60 ± 0.12 mM, L-lactate = 0.82 ± 0.15 mM, pyruvate = 1.03 ± 0.24 mM). Interestingly, hSMCTe oocytes do not display any significant differences of [Na⁺]_i movement for the MCs tested ([Na⁺]_i: butyrate = 0.87 ± 0.23 mM, propionate = 0.83 ± 0.12 mM, L-lactate = 0.69 ± 0.17 mM, pyruvate = 0.85 ± 0.22 mM). This analysis suggests that the affinity of zSMCTe for pyruvate and zSMCTn for pyruvate/L-lactate is different from other MCs.

These results indicate that like human SMCTe, zSMCTe electrogenically transports Na⁺, and Na⁺ transport is elicited by monocarboxylates. Moreover, these results illustrate that zSMCTn is a new electroneutral Na⁺/monocarboxylate cotransporter that is the molecular ortholog of mouse Slc5a12 (32).

Current-Voltage (I-V) Relationship, zSMCT Current—The above data indicate that zSMCTe is electrogenic and the zSMCTn is electroneutral, with both transporting Na⁺ and MCs. We confirmed this difference by measuring currents elicited by Na⁺ and butyrate (1 mM) addition to oocytes expressing zSMCTe (Fig. 3A) or zSMCTn (Fig. 3B). Fig. 3A shows that the zSMCTe oocyte displays a -250 nA current with butyrate addition (open square). Na⁺ removal from the bath solution completely reduced this current (Fig. 3A, open triangle), indicating that butyrate transport is Na⁺-dependent. The zSMCTn oocyte (Fig. 3B) did not show any current change with butyrate addition with or without Na⁺, indicating that the Na⁺ monocarboxylate of zSMCTn (Fig. 2, D and H) is electroneutral. Water-injected controls showed no monocarboxylate-elicited currents (not shown).

Fig. 3C shows the currents (I) elicited from the addition of different monocarboxylates to zSMCTe-expressing oocytes. These currents elicited by addition of 1 mM of each substrate at -150 mV were (in µA) pyruvate =-1.10 ± 0.12, L-lactate = -1.05 ± 0.12, propionate =-1.09 ± 0.15, and butyrate = -1.08 ± 0.16 (n = 6 oocytes in duplicate). Again, the butyrate current was eliminated in the absence of extracellular Na⁺ (average I (µA) at -150 mV: 0 Na⁺ butyrate =-0.14 ± 0.04). The zSMCTe currents induced by different monocarboxylate are similar to hSMCTe currents (I at -150 mV hSMCTe = -1.2 µA) (13, 33).

A recent study by Srinivas et al. (32) reported the cloning of mouse Slc5a12 as a low affinity, electrogenic Na⁺/monocarboxylate cotransporter (SMCT2). Our zSMCTn clone is most closely related to this mouse Slc5a12. Therefore, we tested if zSMCTn might be electrogenic if presented with 40 mM nicotinate (Fig. 3E) or 30 mM L-lactate (Fig. 3G) and compared the zSMCTn responses to those of water-injected control oocytes (Fig. 3, D and F, respectively). We verified that there was no osmolality differences in our test solutions by maintaining constant [Cl^-] and replacing gluconate with lactate or nicotinate. These voltage clamp data indicate that between -150 and +50 mV there is no difference between water-injected and zSMCTn-injected oocytes (not shown), yet Fig. 2, D and H, clearly demonstrates robust changes of [Na⁺]_i.

L-Lactate Transport and Nicotinate Transport—Srinivas et al. (32) reported that they had insufficient protein expression to allow kinetic analysis of mouse Slc5a12. Because we have robust Na⁺ transport mediated by zSMCTn (Slc5a12 ortholog), we compared the affinities of zSMCTn and zSMCTe for L-lactate and nicotinate by measuring either [Na⁺]_i responses (Fig. 4) or current responses (Fig. 5) to varying concentrations. For zSMCTn, Na⁺ intracellular changes during a 5-min exposure (Na⁺_i/5min) to varying concentrations of L-lactate (0–30 mM) or nicotinate (0–50 mM) were measured using Na⁺-selective microelectrodes in a non-voltage-clamped configuration (Fig. 4, A and B). To account for potential biologic variation, we used at least seven zSMCTn-injected oocytes for each MC concentration from at least three different frogs.

View larger version (37K): [in this window] [in a new window]   FIGURE 4. zSMCTn, steady-state kinetics of lactate, nicotinate, and pyruvate transport. Intracellular Na+ concentration ([Na+]i) alterations resulted after addition of various concentrations of L-lactate and nicotinate during a 5-min exposure (A and B). The average [Na+]i/5 min of different concentrations for lactate (A) and nicotinate (B) of at least seven oocytes from three donor animals was fitted to the Michaelis-Menten equation from which the Km values were calculated. C shows the steady-state kinetics for pyruvate from 22Na+ uptake experiments (1 h, see "Experimental Procedures"). D compares the raw 22Na+ uptakes with 2 mM pyruvate for water controls, zSMCTe- and zSMCTn-injected oocytes. Uptake results are from 15 to 20 individual oocytes in each group. Values shown represent mean ± S.E.

View larger version (37K): [in this window] [in a new window]   FIGURE 5. zSMCTe, steady-state kinetics of lactate and nicotinate transport. The currents induced by addition of various concentrations of L-lactate (A) and nicotinate (E) were measured in the presence of 96 mM sodium, whereas the cell was clamped at a series of different membrane potentials. The Michaelis-Menten equation was fit to the current measurements at different membrane potentials for L-lactate (B) and nicotinate (F), from which the derived Imax and Km values are shown in C and D for L-lactate and G and H for nicotinate, respectively. Values shown represent mean ± S.E.; n = 7 oocytes from three donor animals. Substrate concentrations or holding potential (mV) are indicated to the right of the appropriate panel.

Nicotinate Alters L-Lactate Transport by zSMCTn—Under normal physiological circumstances multiple monocarboxylates are present. We were particularly interested in the interrelationship of nicotinate-(K_m 24 mM) and L-lactate (K_m 2 mM)-mediated Na⁺ transport via zSMCTn. To do this, we measured [Na⁺]_i in unclamped zSMCTn oocytes and varied the order of L-lactate and nicotinate addition. Fig. 6A is a representative zSMCTn experiment in which we first exposed a zSMCTn oocyte to 10 mM nicotinate (below K_m) for 5 min and then to 10 mM L-lactate (above K_m). Fig. 6C is a similar zSMCTn experiment in which the oocyte was exposed first to 10 mM L-lactate and then 10 mM nicotinate. In both experiments, L-lactate elicits a large increase of [Na⁺]_i (Fig. 6A, [Na⁺]_i = 1.36 mM, and Fig. 6C, [Na⁺]_i = 2.72 mM). Likewise, 10 mM nicotinate elicited obvious [Na⁺]_i increases (Fig. 6A, [Na⁺]_i = 0.96 mM, and Fig. 6C, [Na⁺]_i = 0.91 mM). The proportion of [Na⁺]_i increase with L-lactate was decreased by 30% after the exposure to nicotinate. The average rates for the "nicotinate-lactate" protocol were 0.15 ± 0.05 mM Na⁺_i/min for nicotinate and 0.33 ± 0.04 mM Na⁺_i/min for lactate. For the "lactate-nicotinate" protocol, the rate of nicotinate-elicited Na⁺_i transport was constant (0.16 ± 0.02 mM Na⁺_i/min) whereas that rate of lactate-elicited [Na⁺]_i transport was dramatically increased (0.57 ± 0.04 mM Na⁺_i/min). Therefore, pre-exposure to L-lactate did not diminish the rate or extent of Na⁺ transport elicited by nicotinate.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. Effect of nicotinate on L-lactate transport in zSMCTn-injected oocytes. Examples of individual experiments of simultaneous measurement of [Na+]i (A and C) and Vm (not shown) for zSMCTn-injected oocytes are shown. Oocytes were continually superfused with 10 mM L-lactate or nicotinate (A, indicated by bars). C, order of perfusion was switched. B and D show the average Na+ transport response ([Na+]i) in the presence of lactate (open bar) and nicotinate (black bar). *, statistical significance (p 0.05) for [Na+]i increase induced by the presence of lactate without previous exposure to nicotinate (D) versus with previous exposure to nicotinate (B). Data represent mean ± S.E. for 6–7 oocytes from at least two frogs.

Ketone Body Transport—Ketone bodies are produced by the liver and used peripherally as an energy source during fasting and other lipolytic stress (1, 2). The two main ketone bodies are acetoacetate (AcAc) and -hydroxybutyrate (OH-buty), whereas acetone is the third, less abundant, ketone body. We therefore evaluated the ability of the zSMCT clones to transport Na⁺ in response to the presence of AcAc and OH-buty (Fig. 7). These keto acids play important roles in basic metabolism (see "Discussion" and Fig. 10A).

Here we show that both zSMCT proteins can transport two of the main ketone bodies (AcAc and OH-buty). Fig. 7 shows individual oocyte experiments simultaneously measuring [Na⁺]_i (top traces) and V_m (bottom traces) after the addition of 1.0 mM AcAc, OH-buty, and butyrate (buty): zSMCTn oocyte (Fig. 7B) and zSMCTe oocyte (Fig. 7C). Both clones in oocytes increase [Na⁺]_i with AcAc (zSMCTn: 2.88–3.71 mM, [Na⁺]_i = 0.83 mM; zSMCTe: 3.31–3.98 mM, [Na⁺]_i = 0.67 mM). Yet a water-injected oocyte (Fig. 7A) does not show any [Na⁺]_i increment (control from 3.23 to 3.16 mM, [Na⁺]_i =-0.07 mM). Only the zSMCTe oocyte showed a [Na⁺]_i increase with the addition of 1.0 mM OH-buty (4.36–5.24 mM, [Na⁺]_i = 0.88 mM). Neither the zSMCTn nor the water-injected oocyte showed a [Na⁺]_i change in the presence of OH-buty (zSMCTn: 3.46 to 3.23 mM, [Na⁺]_i =-0.23 mM; water control, 3.16 to 3.16 mM, [Na⁺]_i = 0.00 mM).

However, the addition of 5 mM OH-buty to a zSMCTn oocyte (Fig. 7H) elicited an obvious [Na⁺]_i increase (2.22–2.76 mM; [Na⁺]_i = 0.54 mM). The same maneuver with the water-injected oocyte (Fig. 7G) did not show a [Na⁺]_i increase (3.78–3.85 mM, [Na⁺]_i = 0.07 mM). Neither oocyte showed a significant V_m change in presence of 5 mM of OH-buty (zSMCTn V_m = 1 mV; water-control V_m = 1 mV). These data suggest that zSMCTn has a lower affinity for OH-buty than zSMCTe. Thus, zSMCTn is also an electroneutral Na⁺/OH-buty cotransporter.

The [Na⁺]_i increases in the presence of ketone bodies and OH-buty for zSMCTe oocytes (Fig. 7C) are coincident with large depolarizations (V_m, AcAc =+23 mV, OH-buty = +34 mV, and butyrate =+59 mV). In contrast, zSMCTn oocytes (Fig. 7B) did not show any V_m change (V_m, AcAc = -1 mV, OH-buty = 0 mV, and butyrate = 0 mV), similar to water-injected oocytes (V_m, AcAc =+1 mV, OH-buty = 0 mV, and butyrate = 0 mV) (Fig. 7A).

The average [Na⁺]_i change ([Na⁺]_i) (Fig. 7, D–F, middle columns) from six zSMCTn oocytes (Fig. 7E) in presence of 1 mM of either ketone body or butyrate show that AcAc ([Na⁺]_i = 1.04 ± 0.06 mM) and butyrate ([Na⁺]_i = 1.11 ± 0.06 mM) are transported with a similar apparent affinity and capacity, but OH-buty ([Na⁺]_i =-0.10 ± 0.06 mM) is not obviously transported at this concentration. In contrast, the average [Na⁺]_i of six zSMCTe oocytes in the presence of 1 mM substrate shows a more obvious difference in the capacity and affinity for both ketone bodies and butyrate (Fig. 7F)([Na⁺]_i: AcAc = 0.25 ± 0.09 mM, OH-buty = 0.50 ± 0.10 mM, and buty = 0.80 ± 0.22 mM). If butyrate-elicited transport is 100%, then Na⁺ transport elicited by AcAc is 31% and OH-buty is 63%. These data are significantly different from the lack of substrate-elicited Na⁺ transport in control oocytes (Fig. 7D; [Na⁺]_i: AcAc =-0.28 ± 0.13, OH-buty =-0.05 ± 0.05, and buty =-0.07 ± 0.03).

View larger version (30K): [in this window] [in a new window]   FIGURE 7. Keto-acid transport by zSMCT, intracellular Na+. Simultaneous measurement of intracellular Na+ concentration ([Na+]i)(top panels) and membrane potential (Vm)(bottom panels) for a water-injected control oocyte (A and G), zSMCTn-injected oocyte (B and H), and zSMCTe-injected oocyte (C). Oocytes were continually superfused as indicated under "Experimental Procedures." 1 mM (A–C) or 5 mM (G and H) of AcAc, OH-buty, or Buty were added as indicated by horizontal lines. Average Na+ transport response ([Na+]i = end[Na+]i - initial [Na+]i) in presence of AcAc (black bar), OH-buty (gray bars), and Buty (open bars) are indicated in the column graphs: water-injected oocytes n = 5(D), zSMCTn-injected oocytes, n = 6(E), and zSMCTe-injected oocytes, n = 6(F). Data were collected from at least two frogs. *, statistical significance (p 0.05) for Na+i increment compared with Buty response for that clone. Individual oocyte experiments are noted with numeric labels (below time bars) indicating the oocyte number used.

In Situ Hybridization—The distribution of zSMCTe and zSMCTn expression was determined by in situ hybridization in zebrafish embryo whole mounts with a digoxigenin-labeled, antisense cRNA probe for zSMCTe and zSMCTn (Fig. 8 and Fig. 9). Control zebrafish embryos show no signal in the organs with reactivity for either zSMCTe or zSMCTn transcripts (Fig. 8, A, D, and G). Signals for zSMCTn and zSMCTe, respectively, were detected in brain and eyes 24 h post-fertilization (hpf) (Fig. 8, B and C), 3 dpf (Fig. 8, E and F), and 5 dpf (Fig. 8, H and I). Both zSMCTe (Fig. 8I) and zSMCTn (Fig. 8H) transcripts are also present in the swim bladder of 5-day-old embryos. zSMCTe (Fig. 8, C, F, and I) and zSMCTn (Fig. 8, E and H) transcripts were also present in the pronephros (embryonic kidney). Although zSMCTn mRNA was in the pronephric tubule (early region of pronephros) (Fig. 8H), zSMCTe mRNA was in pronephric ducts (late region of pronephros) (Fig. 8I). Interestingly, zSMCTe transcripts appeared in the pronephros at 24 hpf (Fig. 8C), whereas zSMCTn transcripts were first evident at 3 dpf (Fig. 8E). We speculate that this message, and presumably protein distribution, is reflective of the absorptive capacity and function as well as the changing role of the pronephros between 24 and 96 hpf.

SLC5A8 was originally cloned from human colon, and thus its expression was expected in the teleost gut. Fig. 8I illustrates that zSMCTe mRNA is present in the gut at 5 dpf.

We further analyzed the zSMCTn and zSMCTe mRNA localization in different tissues by sectioning 5 dpf embryos after whole mount in situ hybridization (Fig. 9). Structures were identified based on the zebrafish anatomy atlas at zebrafish information network (ZFIN). Both SMCT mRNAs were detected in brain (Br), trabecular bar (TB), eyes (E) (Fig. 9, A and E), otic capsule (OC) (Fig. 9, B and F), stomach (S), gallbladder (GB), pronephric duct (PD), pronephric tube (PT) (proximal pronephros) (Fig. 9, C and G), swim bladder (SB), and gut (G) (Fig. 9, D and H).

View larger version (65K): [in this window] [in a new window]   FIGURE 8. Localization of zSMCT mRNA in zebrafish embryos. Whole mount in situ hybridization of embryos at 1 dpf (A–C), 3 dpf (D–F), and 5 dpf (G–I). zSMCTn (B, E, and H) and zSMCTe (C, F, and I) mRNAs are present in the pronephros (pronephric tubule (PT) and pronephric duct (PD)), eye (E), brain (Br), swim bladder (SB), and gut (G)(blue staining and arrows). Note absence of staining in control embryos treated identically, but not exposed to probe (A, D, and G).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We have identified and characterized two Na⁺/monocarboxylate cotransporters from D. rerio (zSMCT), one electrogenic (zSMCTe) and another that is electroneutral (zSMCTn). Molecular and functional characteristics indicate that zSMCTe is the ortholog of hSMCT/SLC5A8. The second transporter is a novel electroneutral SMCT, evidently the 12th member of the Slc5 gene/protein family (zSMCTn/Slc5a12). Like the hSMCT/SLC5A8 cotransporter (13, 33), both zSMCTe and zSMCTn will transport a wide variety of monocarboxylates (short chain fatty acids, pyruvate, lactate, and nicotinate) in a Na⁺-dependent manner.

View larger version (74K): [in this window] [in a new window]   FIGURE 9. Localization of zSMCTn (A–D) and zSMCTe (E–H) in 5 pfd zebrafish embryo sections. 15-µm cryosections of 5 dpf zebrafish embryo whole mounts hybridized in situ with zSMCTn and zSMCTe antisense RNA probes. Transverse sections dorsal to the upper right corner (A and E) or to the top (B–D and F–H). SMCT transcripts (blue staining and arrow) were detected in brain (Br), trabecular bar (TB), eyes (E), otic capsule (OC), stomach (S), gallbladder (GB), pronephric duct (PD), pronephric tubule (PT), swim bladder (SB), gut (G), and exocrine pancreas (P).

View larger version (24K): [in this window] [in a new window]   FIGURE 10. Physiological role of SMCTs in the kidney. A shows a diagram illustrating where the principal substrates of the SMCT transporters enter intermediate metabolism. B illustrates that the proximal tubule reabsorbs MCs via Na+/MCs in two ways: 1Na+:1MC- electroneutral transport by SMCTn (blue) in the earlier proximal tubule where high affinity for MCs is not necessary because uptake occurs, and nNa+:1MC- in the later proximal tube by SMCTe (green) where high affinity and coupling to multiple Na+ ensures absorption.

Li et al. (19) first identified human SLC5A8 as a tumor suppressor in colon cancer. In fact, transfection of colon carcinoma cell lines with SLC5A8 dramatically reduced the growth rate of these cancer cells. This tumor suppressor role appears to be recapitulated in several other epithelial and nervous tissues (20, 37, 38). In support of a differentiation-promoting role, a search for genes involved in early development in Xenopus by Costa et al. (21) identified a transporter sequence Vito, also known as Xenopus Slc5a8, whose mRNA appears early in development (first evident at stage 11 in the superficial ectoderm of the blastopore lip). Vito (Slc5a8) is also abundant at the gastrula and neurulation stages. By stage 35 (organ development), Vito is abundant in the liver diverticulum, the pronephros, and the tail tip (21). Based on their results, these investigators postulated that Vito (two isoforms) is associated with early ectodermal cell determination (21). These data seem consistent with the view that Slc5a8 promotes differentiation. We also predict that demethylation of the Slc5a8 promoter region activates Slc5a8 transcription. Our experiments here have only examined SMCT-mediated transport and SMCT-mRNA localization and thus do not discriminate between these possibilities.

It is well established that apoptosis provides a mechanism to remove damaged or oncogenic cells. It is plausible that restriction of metabolic fuels (lactate, pyruvate, etc.) is another form of cellular defense against growth dysregulation (initial oncogenic step). Slc5a8 (zSMCTe) and Slc5a12 (zSMCTn) are Na⁺-coupled monocarboxylate cotransporters carrying lactate, pyruvate, and other monocarboxylate metabolic fuels across epithelial membranes. Butyrate is a well known inhibitor of histone deacetylases, which directly regulate the extent of winding or unwinding of specific genes on chromosomes (39). Thus, butyrate transport per se may also play a key role in the differentiation or tumor suppressor effects associated with SLC5A8. These cause and effect relationships of monocarboxylate transport, differentiation, and tumor suppression await detailed studies to specifically address these critical issues.

Several studies have also reported the kinetics of renal Na⁺/MC cotransport (7, 8, 12, 34, 40). These studies suggested the presence of two kinetically different Na⁺/MC cotransport systems with one having higher affinity for MCs than the other. In this study we show that zSMCTn has a lower affinity for MCs than zSMCTe or hSMCTe (Figs. 4 and 5). Because zSMCTn is proximal to zSMCTe in the pronephros (see diagram in Fig. 10B), its lower MC affinity is anticipated. The pronephros localizations of zSMCTn and zSMCTe are likely reflective of their physiologic role in the uptake of MCs. The concentration of MCs from the glomerular filtrate that arrives in the early proximal tubule can be high (L-lactate, 1–10 mM in mammals). Thus, efficient absorption of MCs occurs by having a low affinity but high capacity transport of MCs (SMCTn). The MC concentration in the tubule fluid decreases as absorption occurs. Consequently, efficient MC absorption in the later proximal tubule is accomplished by high affinity MC transport (SMCTe) (Fig. 10B).

Studies with salamander proximal tubule showed that luminal, electroneutral lactate transport is Na⁺-coupled and induces an intracellular alkalinization (41), whereas basolateral lactate transport is H⁺-coupled (42). In our experiments we observed a clear Na⁺-dependent, alkalinization mediated by short chain fatty acids (butyrate and propionate) in oocytes injected with zSMCTn. This alkalinization was not observed with the addition of 1 mM pyruvate, lactate, or nicotinate, perhaps because of some endogenous oocyte mechanism for H⁺/pyruvate or H⁺/lactate cotransport that dissipated the alkalinization produced by zSMCTn. In SMCTe- and SMCTn-injected oocytes, alkalinization was consistently elicited by short chain fatty acids in our experiments.

There are 11 SLC5 genes identified in humans (15, 16). Our genomic predictions indicated that a human hypothetical protein MGC52019 (SLC5A12) existed. This sequence is 65% identical to zSMCTn. Recently Srinivas et al. (32) reported a mouse sequence (GenBank^TM AY964639 [GenBank] ; Slc5a12/SMCT2), which is the ortholog of this putative protein. The human chromosomal localization of SLC5A8 (12q22–24) implies that it is a positional gene candidate for association with noninsulin-dependent type 2 diabetes (NIDDM2, MIM 601407 [OMIM] ) (43, 44). It is attractive to speculate that defects in SMCT (SLC5A8) function could cause a saturation of the renal Na⁺-coupled monocarboxylate transport system and might be manifest by monocarboxylates, especially lactate, in the urine. In fact, Thirumurugan et al. (45) have found that L-lactate excretion is increased in the Fanconi syndrome, which encompasses generalized defects in proximal tubule function.

Transport by SMCTs may be the major mechanism by which lipid-modifying drugs gain entry into cells, i.e. clinically useful. Since the 1950s, nicotinic acid (niacin, Niasapan) has been used as a lipid-modifying drug (gram doses) in the treatment of dyslipidemias (46, 47) or dyslipidemia with diabetes (11). Several hypotheses have been proposed regarding the basic mechanism for this niacin effect on blood lipids. These hypotheses include, among other things, a vasodilation effect, a vitamin effect, decreased hepatic cholesterol synthesis, increased cholesterol oxidation, etc. Regardless of the underlying mechanisms, it is clear that the hypolipidemic effect of nicotinate requires the recently identified nicotinate receptor (GPR109A/PUMA-G) (47–49). It is also well established that ligand-receptor interaction time is often determined by ligand degradation (acetylcholine receptors and acetylcholinesterase) or ligand uptake mechanisms. One particularly germane example is vesicular glutamate release at the central nervous system presynaptic terminals, stimulating post-synaptic glutamate receptors and rapid uptake of glutamate by adjacent glutamate transporters (SLC1 family) (50–54). In humans genomic DNA, GPR109A is at 12q24.31 (49) and SLC5A8 is at 12q22–24 (19). It is attractive to speculate that these genes are located in the same region of the genome so that their activities may be coordinated.

Another explanation for the role of nicotinate in dyslipidemia therapy involves inhibition of lipolysis in adipose tissue, resulting in a decreased mobilization of free fatty acids (9–11). Nicotinate is efficiently transported with Na⁺ by both Slc5a8 (zSMCTe) (13, 22) and Slc5a12 (zSMCTn) but with varied affinities and capacities (Figs. 3, 4, 5). Moreover, as illustrated in Fig. 6, the amount and rate of nicotinate transport by zSMCTn are significantly reduced compared with lactate transport. This transport reduction likely points to competition for the monocarboxylate-binding site of zSMCTn such that sterically "bulky" MCs compete with more simple MCs, including short chain fatty acids. These data support the idea that one possible therapeutic action of nicotinic acid in hyperlipidemia is to reduce the absorption of free fatty acid in tissues expressing SMCTs (Slc5a8 and Slc5a12) or MCTs (Slc16a1–a4).

Lactate transport is also reported in brain, but this transport is attributed to H⁺/MC cotransport (MCT, Slc16a1–4) (55, 56). Monocarboxylates such as lactate, pyruvate, and ketone bodies appear to play an important role in brain energy metabolism (Fig. 10A) as well as neuronal and glial intracellular pH buffering (57–59). Lactate has long been considered a waste product disposed of via the circulation; however, when lactate is accumulated it can be toxic for the brain. Monocarboxylates, together with other non-glucose substrates, have long been known to be substantial energy substrates for the developing brain (60). Na⁺/MC cotransport in brain has not been reported previously. Nevertheless, we show clear evidence of both SMCT transcripts in the developing zebrafish brain (Figs. 8 and 9). The roles of the SMCT transporters in the brain and eye remain to be explored, yet central nervous system function of these transporters may provide some clues to early central nervous system development or cellular "programming."

Ketone bodies or keto acids, such as AcAc and OH-buty (Fig. 10A), are always present in the blood, and their levels increase during fasting and prolonged exercise. The metabolic pathway of hepatic ketone body formation (ketogenesis) and extrahepatic ketone body utilization (ketolysis) are especially important for the brain, which uses ketone bodies as a primary energy source when glucose is not available (3). Additionally, ketone bodies are used as substrates for lipid synthesis such as cholesterol for myelin and for neonates and during suckling (1).

Physiologic levels of ketone bodies circulating in plasma range from <0.1 mM (postprandial) to 6 mM (prolonged fasting) and can reach 25 mM in diabetic ketoacidosis. Diabetes is the most common pathological cause of elevated blood keto acids. In diabetic ketoacidosis, high levels of keto acids are produced in response to low insulin levels and high levels of counter-regulatory hormones. In acute diabetic ketoacidosis, the keto acid ratio (OH-buty:AcAc) rises from normal (1:1) to as high as 10:1. In response to insulin therapy, OH-buty levels commonly decrease long before AcAc levels (1–3). The H⁺/MC cotransporters (MCT) are involved in the transport of ketone bodies (4). However, the chromosomal localization of the human homolog of zSMCT (SLC5a8 and SLC5a12) are positional candidates for proteins involved in diseases like noninsulin-dependent type 2 diabetes (NIDDM2 on 12q22–24, MIM 601407 [OMIM] ) (43, 44) and familial combined hyperlipidemia (11p14) (61, 62), respectively.

Our current data suggest that zSMCTe (Slc5a8) and zSMCTn (Slc5a12) have significant differences in affinities for the keto acids AcAc and OH-buty (Fig. 7). Furthermore, these mRNAs and presumably proteins are present in several organs, including the kidney. The chromosome localization of the human SLC5A8 and SLC5A12 orthologs are positional candidates for noninsulin-dependent type II diabetes and hyperlipidemia. We speculate that both SMCTe and SMCTn have important roles in the exacerbating diabetic ketoacidosis because reabsorption of these keto acids by SMCT proteins after glomerular filtration would prevent normal elimination or metabolic use leading to increasing plasma levels of these keto acids and acidosis. Finally, it should be noted that even though zSMCTe is only 50% identical to hSMCT (SLC5A8), the affinity of zSMCTe for lactate and nicotinate were quite similar to those reported for mammalian SMCT (13, 22–24).

In summary, we have identified, cloned, functionally characterized, and localized two distinct Na⁺/monocarboxylate cotransporters in the zebrafish, D. rerio. Our data demonstrate that zSMCTe is the molecular and functional ortholog of human high affinity SMCT (SLC5A8). In the 1st day of zebrafish development, zSMCTe is present, implicating that the presence of SMCT is important for normal development. The second transporter, zSMCTn, is a novel, electroneutral, and low affinity Na⁺/monocarboxylate cotransporter, with a definite mammalian homolog (Slc5a12). Additionally, more than being merely renal or gut transporters, our zebrafish data demonstrate significant expression in the developing brain, eye, and ear. The roles of the SMCT proteins in these tissues will likely lead to novel biochemical, physiologic, and pathophysiologic insights.

	FOOTNOTES

 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY727859 [GenBank] and AY727860 [GenBank] .

^* This work was supported in part by National Institutes of Health Grants DK-56218 (to M. F. R.), DK-60845 (to M. F. R.), and DK-57708 (to D. B. M.), a grant from the Wadsworth Foundation (to C. R. S.), from the American Heart Association Grant SDG06-301037N (to C.R.S.), from the Mexican Council of Science and Technology Grant 48709M (to C.P.), and a postdoctoral fellowship from the American Heart Association Ohio Valley Affiliate (to M.-H. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by National Institutes of Health Grant DK59985 (to R. T. Miller).

² Supported by Veterans Affairs.

³ To whom correspondence should be addressed: Dept. of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, 200 First St. SW, Guggenheim 9-21D, Rochester, MN 55905. Tel.: 507-284-8127; Fax: 484-450-0475; E-mail: romero.michael{at}mayo.edu.

⁴ The abbreviations used are: MCT, monocarboxylate transporter; SMCT, sodium monocarboxylate cotransporter; MCs, monocarboxylates; [Na⁺]_i, intracellular Na⁺ concentration; AcAc, acetoacetate; SMCTe, electrogenic; SMCTn, electroneutral; EST, expressed sequence tag; AP, alkaline phosphatase; OH-buty, -hydroxybutyrate; hpf, hours post-fertilization; dpf, days post-fertilization.

⁵ Several MCs were added and washed out in each experiment. The order of additions was randomized. Each MC solution bathed the oocyte for 5 min. These results did not differ significantly from experiments with a single MC addition. ²²Na⁺ uptake experiments revealed that 1 mM MC elicited a linear accumulation between 5 and 90 min (not shown).

⁶ The [Na⁺]_i response of zSMCTn-injected oocytes to MCs was linear over at least the first 5 min.

⁷ The average unclamped voltages for zSMCTn oocytes (Fig. 4) for these experiments was -45 ± 6 mV. Thus, a comparison of the zSNCTn-K_m values to the zSMCTe-K_m values at -50 mV (Fig. 5) was used to calculate the 10- or 30-fold difference.

	ACKNOWLEDGMENTS

 
We thank Montelle Sanders and Gerald Babcock for excellent technical support. We also thank Dr. R. T. Miller and H. L. Holmes for helpful comments on the manuscript. We especially thank Dr. Iain Drummond (Renal Unit, Massachusetts General Hospital and Harvard) for insights and clarification of the appearance of mesonephric ducts and tubules in zebrafish.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Geibisch, G., and Windhager, E. (2003) in Medical Physiology (Boron, W. F., and Boulpaep, E. L., eds) 1st Ed., pp. 790-813, W. B. Saunders Co., Philadelphia
2. Juel, C., and Halestrap, A. P. (1999) J. Physiol. (Lond.) 517, 633-642[Abstract/Free Full Text]
3. Halestrap, A. P., and Meredith, D. (2004) Pfluegers Arch. 447, 619-628[CrossRef][Medline] [Order article via Infotrieve]
4. Barbarat, B., and Podevin, R. A. (1988) J. Biol. Chem. 263, 12190-12193[Abstract/Free Full Text]
5. Siebens, A. W., and Boron, W. F. (1987) J. Gen. Physiol. 90, 799-831[Abstract/Free Full Text]
6. Barac-Nieto, M., Murer, H., and Kinne, R. (1980) Am. J. Physiol. 239, F496-F506[Medline] [Order article via Infotrieve]
7. Mengual, R., Claude-Schlageter, M. H., Poiree, J. C., Yagello, M., and Sudaka, P. (1989) J. Membr. Biol. 108, 197-205[CrossRef][Medline] [Order article via Infotrieve]
8. Mengual, R., Schlageter, M. H., and Sudaka, P. (1990) J. Biol. Chem. 265, 292-299[Abstract/Free Full Text]
9. Carlson, L. A. (2005) J. Intern. Med. 258, 94-114[CrossRef][Medline] [Order article via Infotrieve]
10. Carlson, L. A. (2004) Int. J. Clin. Pract. 58, 706-713[CrossRef][Medline] [Order article via Infotrieve]
11. Shepherd, J., Betteridge, J., and Van Gaal, L. (2005) Curr. Med. Res. Opin. 21, 665-682[CrossRef][Medline] [Order article via Infotrieve]
12. Manganel, M., Roch-Ramel, F., and Murer, H. (1985) Am. J. Physiol. 249, F400-F408[Medline] [Order article via Infotrieve]
13. Coady, M. J., Chang, M.-H., Charron, F. M., Plata, C., Wallendorff, B., Sah, J. F., Markowitz, S. D., Romero, M. F., and Lapointe, J.-Y. (2004) J. Physiol. (Lond.) 557, 719-731[Abstract/Free Full Text]
14. Miyauchi, S., Gopal, E., Fei, Y. J., and Ganapathy, V. (2004) J. Biol. Chem. 279, 13293-13296[Abstract/Free Full Text]
15. Wright, E. M., and Turk, E. (2004) Pfluegers Arch. 447, 510-518[CrossRef][Medline] [Order article via Infotrieve]
16. Wright, E. M., Loo, D. D., Hirayama, B. A., and Turk, E. (2004) Physiology (Bethesda) 19, 370-376[CrossRef][Medline] [Order article via Infotrieve]
17. Rodriguez, A. M., Perron, B., Lacroix, L., Caillou, B., Leblanc, G., Schlumberger, M., Bidart, J. M., and Pourcher, T. (2002) J. Clin. Endocrinol. Metab. 87, 3500-3503[Abstract/Free Full Text]
18. Dai, G., Levy, O., and Carrasco, N. (1996) Nature 379, 458-460[CrossRef][Medline] [Order article via Infotrieve]
19. Li, H., Myeroff, L., Smiraglia, D., Romero, M. F., Pretlow, T., Kasturi, L., Lutterbaugh, J., Casey, G., Issa, J.-P., Willis, J., Willson, J. K. V., Plass, C., and Markowitz, S. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 8412-8417[Abstract/Free Full Text]
20. Porra, V., Ferraro-Peyret, C., Durand, C., Selmi-Ruby, S., Giroud, H., Berger-Dutrieux, N., Decaussin, M., Peix, J. L., Bournaud, C., Orgiazzi, J., Borson-Chazot, F., Dante, R., and Rousset, B. (2005) J. Clin. Endocrinol Metab. 90, 3028-3035[Abstract/Free Full Text]
21. Costa, R. M., Mason, J., Lee, M., Amaya, E., and Zorn, A. M. (2003) Gene Expr. Patterns 3, 509-519[CrossRef][Medline] [Order article via Infotrieve]
22. Gopal, E., Fei, Y. J., Miyauchi, S., Zhuang, L., Prasad, P. D., and Ganapathy, V. (2005) Biochem. J. 388, 309-316[CrossRef][Medline] [Order article via Infotrieve]
23. Gopal, E., Fei, Y. J., Sugawara, M., Miyauchi, S., Zhuang, L., Martin, P., Smith, S. B., Prasad, P. D., and Ganapathy, V. (2004) J. Biol. Chem. 279, 44522-44532[Abstract/Free Full Text]
24. Ganapathy, V., Gopal, E., Miyauchi, S., and Prasad, P. D. (2005) Biochem. Soc. Trans. 33, 237-240[CrossRef][Medline] [Order article via Infotrieve]
25. Sciortino, C. M., and Romero, M. F. (1999) Am. J. Physiol. 277, F611-F623[Medline] [Order article via Infotrieve]
26. Romero, M. F., Fong, P., Berger, U. V., Hediger, M. A., and Boron, W. F. (1998) Am. J. Physiol. 274, F425-F432[Medline] [Order article via Infotrieve]
27. Romero, M. F., Henry, D., Nelson, S., Harte, P. J., Dillon, A. K., and Sciortino, C. M. (2000) J. Biol. Chem. 275, 24552-24559[Abstract/Free Full Text]
28. Romero, M. F., Hediger, M. A., Boulpaep, E. L., and Boron, W. F. (1997) Nature 387, 409-413[CrossRef][Medline] [Order article via Infotrieve]
29. Dinour, D., Chang, M.-H., Satoh, J.-I., Smith, B. L., Angle, N., Knecht, A., Serban, I., Holtzman, E. J., and Romero, M. F. (2004) J. Biol. Chem. 279, 52238-52246[Abstract/Free Full Text]
30. Plata, C., Meade, P., Vazquez, N., Hebert, S. C., and Gamba, G. (2002) J. Biol. Chem. 277, 11004-11012[Abstract/Free Full Text]
31. Thisse, C., Thisse, B., Schilling, T. F., and Postlethwait, J. H. (1993) Development (Camb.) 119, 1203-1215[Abstract]
32. Srinivas, S. R., Gopal, E., Zhuang, L., Itagaki, S., Martin, P. M., Fei, Y. J., Ganapathy, V., and Prasad, P. D. (2005) Biochem. J. 392, 655-664[CrossRef][Medline] [Order article via Infotrieve]
33. Chang, M.-H., Plata, C., and Romero, M. F. (2004) J. Am. Soc. Nephrol. 15, 75 (abstr.)
34. Jorgensen, K. E., and Sheikh, M. I. (1986) Biochim. Biophys. Acta 860, 632-640[Medline] [Order article via Infotrieve]
35. Yoon, H., Fanelli, A., Grollman, E. F., and Philp, N. J. (1997) Biochem. Biophys. Res. Commun. 234, 90-94[CrossRef][Medline] [Order article via Infotrieve]
36. Gerhart, D. Z., Leino, R. L., and Drewes, L. R. (1999) Neuroscience 92, 367-375[CrossRef][Medline] [Order article via Infotrieve]
37. Ueno, M., Toyota, M., Akino, K., Suzuki, H., Kusano, M., Satoh, A., Mita, H., Sasaki, Y., Nojima, M., Yanagihara, K., Hinoda, Y., Tokino, T., and Imai, K. (2004) Tumor Biol. 25, 134-140[CrossRef]
38. Hong, C., Maunakea, A., Jun, P., Bollen, A. W., Hodgson, J. G., Goldenberg, D. D., Weiss, W. A., and Costello, J. F. (2005) Cancer Res. 65, 3617-3623[Abstract/Free Full Text]
39. Davie, J. R. (2003) J. Nutr. 133, S2485-S2493[Abstract/Free Full Text]
40. Nord, E., Wright, S. H., Kippen, I., and Wright, E. M. (1982) Am. J. Physiol. 243, F456-F462[Medline] [Order article via Infotrieve]
41. Siebens, A. W., and Boron, W. F. (1989) Am. J. Physiol. 256, F342-F353[Medline] [Order article via Infotrieve]
42. Siebens, A. W., and Boron, W. F. (1989) Am. J. Physiol. 256, F354-F365[Medline] [Order article via Infotrieve]
43. Lindgren, C. M., Mahtani, M. M., Widen, E., McCarthy, M. I., Daly, M. J., Kirby, A., Reeve, M. P., Kruglyak, L., Parker, A., Meyer, J., Almgren, P., Lehto, M., Kanninen, T., Tuomi, T., Groop, L. C., and Lander, E. S. (2002) Am. J. Hum. Genet. 70, 509-516[CrossRef][Medline] [Order article via Infotrieve]
44. Frayling, T. M., Lindgren, C. M., Chevre, J. C., Menzel, S., Wishart, M., Benmezroua, Y., Brown, A., Evans, J. C., Rao, P. S., Dina, C., Lecoeur, C., Kanninen, T., Almgren, P., Bulman, M. P., Wang, Y., Mills, J., Wright-Pascoe, R., Mahtani, M. M., Prisco, F., Costa, A., Cognet, I., Hansen, T., Pedersen, O., Ellard, S., Tuomi, T., Groop, L. C., Froguel, P., Hattersley, A. T., and Vaxillaire, M. (2003) Diabetes 52, 872-881[Abstract/Free Full Text]
45. Thirumurugan, A., Thewles, A., Gilbert, R. D., Hulton, S. A., Milford, D. V., Lote, C. J., and Taylor, C. M. (2004) Nephrol. Dial. Transplant. 19, 1767-1773[Abstract/Free Full Text]
46. Pike, N. B., and Wise, A. (2004) Curr. Opin. Investig. Drugs 5, 271-275[Medline] [Order article via Infotrieve]
47. Tunaru, S., Kero, J., Schaub, A., Wufka, C., Blaukat, A., Pfeffer, K., and Offermanns, S. (2003) Nat. Med. 9, 352-355[CrossRef][Medline] [Order article via Infotrieve]
48. Taggart, A. K., Kero, J., Gan, X., Cai, T. Q., Cheng, K., Ippolito, M., Ren, N., Kaplan, R., Wu, K., Wu, T. J., Jin, L., Liaw, C., Chen, R., Richman, J., Connolly, D., Offermanns, S., Wright, S. D., and Waters, M. G. (2005) J. Biol. Chem. 280, 26649-26652[Abstract/Free Full Text]
49. Wise, A., Foord, S. M., Fraser, N. J., Barnes, A. A., Elshourbagy, N., Eilert, M., Ignar, D. M., Murdock, P. R., Steplewski, K., Green, A., Brown, A. J., Dowell, S. J., Szekeres, P. G., Hassall, D. G., Marshall, F. H., Wilson, S., and Pike, N. B. (2003) J. Biol. Chem. 278, 9869-9874[Abstract/Free Full Text]
50. Mitani, A., and Tanaka, K. (2003) J. Neurosci. 23, 7176-7182[Abstract/Free Full Text]
51. Kanai, Y., Bhide, P. G., DiFiglia, M., and Hediger, M. A. (1995) Neuroreport 6, 2357-2362[Medline] [Order article via Infotrieve]
52. Kanai, Y., Nussberger, S., Romero, M. F., Boron, W. F., Hebert, S. C., and Hediger, M. A. (1995) J. Biol. Chem. 270, 16561-16568[Abstract/Free Full Text]
53. Rothstein, J. D., Dykes-Hoberg, M., Pardo, C. A., Bristol, L. A., Jin, L., Kuncl, R. W., Kanai, Y., Hediger, M. A., Wang, Y., Schielke, J. P., and Welty, D. F. (1996) Neuron 16, 675-686[CrossRef][Medline] [Order article via Infotrieve]
54. Furuta, A., Noda, M., Suzuki, S. O., Goto, Y., Kanahori, Y., Rothstein, J. D., and Iwaki, T. (2003) Am. J. Pathol. 163, 779-787[Abstract/Free Full Text]
55. Soengas, J. L., and Aldegunde, M. (2002) Comp. Biochem. Physiol. B Biochem. Mol. Biol. 131, 271-296[CrossRef][Medline] [Order article via Infotrieve]
56. Hertz, L., and Dienel, G. A. (2005) J. Neurosci. Res. 79, 11-18[CrossRef][Medline] [Order article via Infotrieve]
57. Deitmer, J. W. (2002) J. Neurochem. 80, 721-726[CrossRef][Medline] [Order article via Infotrieve]
58. Becker, H. M., Broer, S., and Deitmer, J. W. (2004) Biophys. J. 86, 235-247[Medline] [Order article via Infotrieve]
59. Becker, H. M., and Deitmer, J. W. (2004) J. Biol. Chem. 279, 28057-28062[Abstract/Free Full Text]
60. Pierre, K., and Pellerin, L. (2005) J. Neurochem. 94, 1-14[Medline] [Order article via Infotrieve]
61. Shoulders, C. C., Jones, E. L., and Naoumova, R. P. (2004) Hum. Mol. Genet. 13, Suppl. 1, R149-R160[Abstract/Free Full Text]
62. Naoumova, R. P., Bonney, S. A., Eichenbaum-Voline, S., Patel, H. N., Jones, B., Jones, E. L., Amey, J., Colilla, S., Neuwirth, C. K., Allotey, R., Seed, M., Betteridge, D. J., Galton, D. J., Cox, N. J., Bell, G. I., Scott, J., and Shoulders, C. C. (2003) Arterioscler. Thromb. Vasc. Biol. 23, 2070-2077[Abstract/Free Full Text]

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