SLC5A8, a tumor suppressor gene down-regulated in human colon cancer, codes for a transporter in the Na؉ / glucose cotransporter gene family, but the definitive functional identity of the transporter protein is not known. Since this gene is expressed abundantly in the colon where short-chain fatty acids are generated by bacterial fermentation, we tested the hypothesis that it codes for a Na ؉ -coupled transporter for these fatty acids. The coding region of SLC5A8 mRNA was amplified from human intestine and expressed heterologously in Xenopus laevis oocytes. Transport function was monitored by uptake of radiolabeled substrates and by substrate-induced currents under voltage-clamp conditions. Uptake of short-chain fatty acids (lactate, pyruvate, acetate, propionate, and butyrate) in oocytes expressing SLC5A8 was severalfold higher than in uninjected oocytes. Exposure of SLC5A8-expressing oocytes to these fatty acids induced inward currents under voltageclamp conditions in a Na ؉ -dependent manner. These currents were saturable and the substrate concentrations needed for half-maximal induction of the current were in the range of 0.08 -2.5 mM. The substrate-induced currents decreased as the carbon chain length of the substrates increased. The Na ؉ -activation kinetics indicated involvement of more than one Na ؉ ion in the activation process. Direct measurements of substrate (propionate) and charge transfer showed that three positive charges are transferred into oocytes per substrate molecule. These studies establish the functional identity of SLC5A8 as a Na ؉ -coupled transporter for short-chain fatty acids.

Abstract. SLC5A8 and SLC5A12 are sodium-coupled monocarboxylate transporters (SMCTs), the former being a high-affinity type and the latter a low-affinity type. Both transport a variety of monocarboxylates in a Na + -coupled manner. They are expressed in the gastrointestinal tract, kidney, thyroid, brain, and retina. SLC5A8 is localized to the apical membrane of epithelial cells lining the intestinal tract and proximal tubule. In the brain and retina, its expression is restricted to neurons and the retinal pigment epithelium. The physiologic functions of SLC5A8 include absorption of short-chain fatty acids in the colon and small intestine, reabsorption of lactate and pyruvate in the kidney, and cellular uptake of lactate and ketone bodies in neurons. It also transports the B-complex vitamin nicotinate. SLC5A12 is also localized to the apical membrane of epithelial cells lining the intestinal tract and proximal tubule. In the brain and retina, its expression is restricted to astrocytes and Müller cells. SLC5A8 also functions as a tumor suppressor; its expression is silenced in tumors of colon, thyroid, stomach, kidney, and brain. The tumor-suppressive function is related to its ability to mediate concentrative uptake of butyrate, propionate, and pyruvate, all of which are inhibitors of histone deacetylases. SLC5A8 can also transport a variety of pharmacologically relevant monocarboxylates, including salicylates, benzoate, and g-hydroxybutyrate. Non-steroidal anti-inflammatory drugs such as ibuprofen, ketoprofen, and fenoprofen, also interact with SLC5A8. These drugs are not transportable substrates for SLC5A8, but instead function as blockers of the transporter. Relatively less is known on the role of SLC5A12 in drug transport.

Tumor cells up-regulate glycolysis but convert pyruvate into lactate instead of oxidizing it. Here, we show that pyruvate, but not lactate, is an inhibitor of histone deacetylases (HDAC) and an inducer of apoptosis in tumor cells and that SLC5A8, a Na + /monocarboxylate cotransporter, is obligatory for this process. We found that SLC5A8 is expressed in nontransformed breast epithelial cell lines but silenced by DNA methylation in tumor cell lines. The down-regulation of the gene is also evident in primary breast tumors. When MCF7 breast tumor cells are transfected with SLC5A8 cDNA, the cells undergo pyruvate-dependent apoptosis. Butyrate and propionate also induce apoptosis in SLC5A8-expressing cells, whereas lactate does not. The differential ability of these monocarboxylates to cause apoptosis in SLC5A8-expressing MCF7 cells correlates with their ability to inhibit HDACs. Apoptosis induced by SLC5A8/pyruvate in MCF7 cells is associated with up-regulation of p53, Bax, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), TRAIL receptor (TRAILR) 1, and TRAILR2 and down-regulation of Bcl2 and survivin. Lactate dehydrogenase isozymes are differentially expressed in nontransformed cells and tumor cells such that the latter convert pyruvate into lactate. Silencing of SLC5A8 coupled with conversion of pyruvate into lactate in tumor cells correlates with increased HDAC activity in these cells compared with nontransformed cells. Our studies thus identify pyruvate as a HDAC inhibitor and indicate that the Na + -coupled pyruvate transport underlies the tumor-suppressive role of SLC5A8. We propose that tumor cells silence SLC5A8 and convert pyruvate into lactate as complementary mechanisms to avoid pyruvate-induced cell death.

We report here on the expression of slc5a8 in kidney and its relevance to Na ؉ -coupled reabsorption of lactate. slc5a8 is the murine ortholog of SLC5A8, a candidate tumor suppressor gene, which we recently cloned from human intestine and demonstrated its functional identity as a Na ؉ -coupled transporter for short-chain fatty acids and lactate. The slc5a8 cDNA, cloned from mouse kidney, codes for a protein consisting of 611 amino acids. When expressed heterologously in mammalian cells or Xenopus oocytes, slc5a8 mediates Na ؉ -coupled electrogenic transport of lactate/pyruvate as well as short-chain fatty acids (e.g. acetate, propionate, and butyrate). The Na ؉ /fatty acid stoichiometry varies depending on the fatty acid substrate (2:1 for lactate and 4:1 for propionate). This phenomenon of variable Na ؉ / substrate stoichiometry depending on the fatty acid substrate is also demonstrable with human SLC5A8. In situ hybridization with sagittal sections of mouse kidney demonstrates abundant expression of the transcripts in the cortex as well as the medulla. Brush border membrane vesicles prepared from rabbit kidney are able to transport lactate in a Na ؉ -coupled manner. The transport process exhibits the overshoot phenomenon, indicating uphill lactate transport in response to the transmembrane Na ؉ gradient. The Na ؉ -coupled lactate transport in these membrane vesicles is inhibitable by short-chain fatty acids. We conclude that slc5a8 is expressed abundantly in the kidney and that it plays a role in the active reabsorption of lactate. slc5a8 is the first transporter known to be expressed in mammalian kidney that has the ability to mediate the Na ؉ -coupled reabsorption of lactate.L-Lactate is present in blood at a concentration of ϳ90 mg/ liter (ϳ1.5 mM), but the urinary excretion of L-lactate is very low (100 -600 mg/day). With the normal glomerular filtration rate of 120 ml/min, the fractional reabsorption rate for L-lactate in mammalian kidney is Ͼ95% (1). The molecular identity of the transport system that is responsible for such an effective absorption process in the kidney has not yet been established. L-Lactate transport across mammalian cell plasma membrane is mediated by monocarboxylate transporters (MCTs) 1 (2, 3).MCTs are H ϩ -coupled transporters and, therefore, the direction of lactate flux in mammalian cells depends on the net chemical gradients for H ϩ and lactate across the membrane. The transport process is electroneutral because of the H ϩ / lactate stoichiometry of 1:1. There are several members within the MCT gene family that are expressed differentially in different tissues (2, 3). Many of the MCT gene family members are expressed in the kidney (2-5), but the exact locations of these transporters in terms of cell type and the apical membrane versus the basolateral membrane of the tubular cells are not known. Because the lumen-facing brush border membrane of the renal tubular cells mediates the first step in the reabsorption of solutes present in the glomerular filtrate, studies have been carr...

In the present study, we report on the molecular cloning and functional characterization of mouse NaCT (Na+-coupled citrate transporter), the mouse orthologue of Drosophila Indy. Mouse NaCT consists of 572 amino acids and is highly similar to rat and human NaCTs in primary sequence. The mouse nact gene coding for the transporter is approx. 23 kb long and consists of 12 exons. When expressed in mammalian cells, the cloned transporter mediates the Na+-coupled transport of citrate and succinate. Competition experiments reveal that mouse NaCT also recognizes other tricarboxylic acid cycle intermediates such as malate, fumarate and 2-oxo-glutarate as excellent substrates. The Michaelis-Menten constant for the transport process is 38+/-5 mM for citrate and 37+/-6 mM for succinate at pH 7.5. The transport process is electrogenic and exhibits an obligatory requirement for Na+. Na+-activation kinetics indicates that multiple Na+ ions are involved in the activation process. Extracellular pH has a differential effect on the transport function of mouse NaCT depending on whether the transported substrate is citrate or succinate. The Michaelis-Menten constants for these substrates are also influenced markedly by pH. When examined in the Xenopus laevis oocyte expression system with the two-microelectrode voltage-clamp technique, the transport process mediated by mouse NaCT is electrogenic. The charge-to-substrate ratio is 1 for citrate and 2 for succinate. The most probable transport mechanism predicted by these studies involves the transport of citrate as a tervalent anion and succinate as a bivalent anion with a fixed Na+/substrate stoichiometry of 4:1. The present study provides the first unequivocal evidence for the electrogenic nature of mammalian NaCT.

We report in the present paper, on the isolation and functional characterization of slc5a12, the twelfth member of the SLC5 gene family, from mouse kidney. The slc5a12 cDNA codes for a protein of 619 amino acids. Heterologous expression of slc5a12 cDNA in mammalian cells induces Na+-dependent transport of lactate and nicotinate. Several other short-chain monocarboxylates compete with nicotinate for the cDNA-induced transport process. Expression of slc5a12 in Xenopus oocytes induces electrogenic and Na+-dependent transport of lactate, nicotinate, propionate and butyrate. The substrate specificity of slc5a12 is similar to that of slc5a8, an Na+-coupled transporter for monocarboxylates. However, the substrate affinities of slc5a12 were much lower than those of slc5a8. slc5a12 mRNA is expressed in kidney, small intestine and skeletal muscle. In situ hybridization with sagittal sections of mouse kidney showed predominant expression of slc5a12 in the outer cortex. This is in contrast with slc5a8, which is expressed in the cortex as well as in the medulla. The physiological function of slc5a12 in the kidney is likely to mediate the reabsorption of lactate. In the intestinal tract, slc5a12 is expressed in the proximal parts, whereas slc5a8 is expressed in the distal parts. The expression of slc5a12 in the proximal parts of the intestinal tract, where there is minimal bacterial colonization, suggests that the physiological function of slc5a12 is not to mediate the absorption of short-chain monocarboxylates derived from bacterial fermentation but rather to mediate the absorption of diet-derived short-chain monocarboxylates. Based on the functional and structural similarities between slc5a8 and slc5a12, we suggest that the two transporters be designated as SMCT1 (sodium-coupled monocarboxylate transporter 1) and SMCT2 respectively.

SMCT1 is a sodium-coupled (Na + -coupled) transporter for L-lactate and short-chain fatty acids. Here, we show that the ketone bodies, b-D-hydroxybutyrate and acetoacetate, and the branched-chain ketoacid, a-ketoisocaproate, are also substrates for the transporter. The transport of these compounds via human SMCT1 is Na + -coupled and electrogenic. The Michaelis constant is 1.4 ± 0.1 mM for b-D-hydroxybutyrate, 0.21 ± 0.04 mM for acetoacetate and 0.21 ± 0.03 mM for a-ketoisocaproate. The Na + : substrate stoichiometry is 2 : 1.As L-lactate and ketone bodies constitute primary energy substrates for neurons, we investigated the expression pattern of this transporter in the brain.In situ hybridization studies demonstrate widespread expression of SMCT1 mRNA in mouse brain. Immunofluorescence analysis shows that SMCT1 protein is expressed exclusively in neurons. SMCT1protein co-localizes with MCT2, a neuron-specific Na + -independent monocarboxylate transporter. In contrast, there was no overlap of signals for SMCT1 and MCT1, the latter being expressed only in non-neuronal cells. We also demonstrate the neuron-specific expression of SMCT1 in mixed cultures of rat cortical neurons and astrocytes. This represents the first report of an Na + -coupled transport system for a major group of energy substrates in neurons. These findings suggest that SMCT1 may play a critical role in the entry of L-lactate and ketone bodies into neurons by a process driven by an electrochemical Na + gradient and hence, contribute to the maintenance of the energy status and function of neurons. Keywords: brain, ketone bodies, neuroenergetics, neuronspecific expression, sodium-coupled monocarboxylate transporter, sodium-coupled transport. Glucose is the major energy substrate used by the brain (Pellerin and Magistretti 2004). The metabolism of glucose in the brain is compartmentalized. Even though the metabolic utilization of glucose in the brain is increased during intense neuronal activity, uptake of glucose in astrocytes, rather than in neurons, accounts for most of this activity-associated glucose utilization (Pellerin and Magistretti 2004). Astrocytes convert glucose into L-lactate, a monocarboxylate, and release it into the extracellular medium to be taken up subsequently by neurons. Thus, the energy needs of the active neurons are not met directly by glucose oxidation but rather, by oxidation of L-lactate supplied by astrocytes. L-Lactate is therefore the primary metabolic fuel for neurons under normal physiological conditions. Normal levels of L-lactate in blood are quite significant (1-1.2 mM) and this L-lactate in the circulation is also available for neurons, in addition to the L-lactate generated by astrocytes.Under circumstances when glucose availability is limited, such as during the suckling period in mammals (Hawkins et al. 1971;Cremer 1982), prolonged starvation and uncontrolled diabetes (Gjedde and Crone 1975;Hawkins et al. 1986), neurons must rely on substrates other than L-lactate to fulfil their high energy demands a...

In this article, we report on the expression and function of a Na(+)-coupled transporter for citrate, NaCT, in human and rat liver cell lines and in primary hepatocytes from the rat liver. We also describe the polarized expression of this transporter in human and rat livers. Citrate uptake in human liver cell lines HepG2 and Huh-7 was obligatorily dependent on Na+. The uptake system showed a preference for citrate over other intermediates of the citric acid cycle and exhibited a Michaelis constant of approximately 6 mM for citrate. The transport activity was stimulated by Li+, and the activation was associated with a marked increase in substrate affinity. Citrate uptake in rat liver cell line MH1C1 was also Na+ dependent and showed a preference for citrate. The Michaelis constant for citrate was approximately 10 microM. The transport activity was inhibited by Li+. Primary hepatocytes from the rat liver also showed robust activity for Na+-coupled citrate uptake, with functional features similar to those described in the rat liver cell line. Immunolabeling with a specific anti-NaCT antibody showed exclusive expression of the transporter in the sinusoidal membrane of hepatocytes in human and rat livers. This constitutes the first report on the expression and function of NaCT in liver cells.