Seiji Miyauchi

Sodium-coupled Monocarboxylate Transporters in Normal Tissues and in Cancer

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 γ-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.

Structure, function and immunolocalization of a proton‐coupled amino acid transporter (hPAT1) in the human intestinal cell line Caco‐2

The human orthologue of the H+‐coupled amino acid transporter (hPAT1) was cloned from the human intestinal cell line Caco‐2 and its functional characteristics evaluated in a mammalian cell heterologous expression system. The cloned hPAT1 consists of 476 amino acids and exhibits 85 % identity with rat PAT1. Among the various human tissues examined by Northern blot, PAT1 mRNA was expressed most predominantly in the intestinal tract. When expressed heterologously in mammalian cells, hPAT1 mediated the transport of α‐(methylamino)isobutyric acid (MeAIB). The cDNA‐induced transport was Na+‐independent, but was energized by an inwardly directed H+ gradient. hPAT1 interacted with glycine, l‐alanine, l‐proline, α‐aminoisobutyrate (AIB) and γ‐aminobutyrate (GABA), as evidenced from direct transport measurements and from competition experiments with MeAIB as a transport substrate. hPAT1 also recognized the d‐isomers of alanine and proline. With serine and cysteine, though the l‐isomers did not interact with hPAT1 to any significant extent, the corresponding d‐isomers were recognized as substrates. With proline and alanine, the affinity was similar for l‐ and d‐isomers. However, with cysteine and serine, the d‐isomers showed 6‐ to 8‐fold higher affinity for hPAT1 than the corresponding l‐isomers. These functional characteristics of hPAT1 closely resemble those that have been described previously for the H+‐coupled amino acid transport system in Caco‐2 cells. Furthermore, there was a high degree of correlation (r2= 0.93) between the relative potencies of various amino acids to inhibit the H+‐coupled MeAIB transport measured with native Caco‐2 cells and with hPAT1 in the heterologous expression system. Immunolocalization studies showed that PAT1 was expressed exclusively in the apical membrane of Caco‐2 cells. These data suggest that hPAT1 is responsible for the H+‐coupled amino acid transport expressed in the apical membrane of Caco‐2 cells.

Transport of Nicotinate and Structurally Related Compounds by Human SMCT1 (SLC5A8) and Its Relevance to Drug Transport in the Mammalian Intestinal Tract

PurposeTo examine the involvement of human SMCT1, a Na+-coupled transporter for short-chain fatty acids, in the transport of nicotinate/structural analogs and monocarboxylate drugs, and to analyze its expression in mouse intestinal tract.Materials and MethodsWe expressed human SMCT1 in X. laevis oocytes and monitored its function by [14C]nicotinate uptake and substrate-induced inward currents. SMCT1 expression in mouse intestinal tract was examined by immunofluorescence.Results[14C]Nicotinate uptake was several-fold higher in SMCT1-expressing oocytes than in water-injected oocytes. The uptake was inhibited by short-chain/medium-chain fatty acids and various structural analogs of nicotinate. Exposure of SMCT1-expressing oocytes to nicotinate induced Na+-dependent inward currents. Measurements of nicotinate flux and associated charge transfer into oocytes suggest a Na+:nicotinate stoichiometry of 2:1. Monocarboxylate drugs benzoate, salicylate, and 5-aminosalicylate are also transported by human SMCT1. The transporter is expressed in the small intestine as well as colon, and the expression is restricted to the lumen-facing apical membrane of intestinal and colonic epithelial cells.ConclusionsHuman SMCT1 transports not only nicotinate and its structural analogs but also various monocarboxylate drugs. The transporter is expressed on the luminal membrane of the epithelial cells lining the intestinal tract. SMCT1 may participate in the intestinal absorption of monocarboxylate drugs.

Biological functions of SLC5A8, a candidate tumour suppressor

SLC5A8 is a candidate tumour suppressor gene that is silenced in colon cancer, gastric cancer and possibly other cancers in humans. This gene codes for a transporter belonging to the Na(+)/glucose co-transporter gene family (SLC5). The cancer-associated silencing of the gene involves hypermethylation of CpG islands present in exon 1 of the gene. SLC5A8 is expressed in colon, ileum, kidney and thyroid gland. The protein coded by the gene mediates the Na(+)-coupled and electrogenic transport of a variety of monocarboxylates, including short-chain fatty acids, lactate and nicotinate. It may also transport iodide. The normal physiological function of this transporter in the intestinal tract and kidney is likely to facilitate the active absorption of short-chain fatty acids, lactate and nicotinate. One of the short-chain fatty acids that serves as a substrate for SLC5A8 is butyrate. This fatty acid is an inhibitor of histone deacetylases and is known to induce apoptosis in a variety of tumours including colonic tumour. Since butyrate is produced in the colonic lumen at high concentrations by bacterial fermentation of dietary fibre, we speculate that the ability of SLC5A8 to mediate the entry of this short-chain fatty acid into colonic epithelial cells underlies the potential tumour suppressor function of this transporter.

Relevance of NAC-2, an Na+-coupled citrate transporter, to life span, body size and fat content in Caenorhabditis elegans.

We have cloned and functionally characterized an Na+-coupled citrate transporter from Caenorhabditis elegans (ceNAC-2). This transporter shows significant sequence homology to Drosophila Indy and the mammalian Na+-coupled citrate transporter NaCT (now known as NaC2). When heterologously expressed in a mammalian cell line or in Xenopus oocytes, the cloned ceNAC-2 mediates the Na+-coupled transport of various intermediates of the citric acid cycle. However, it transports the tricarboxylate citrate more efficiently than dicarboxylates such as succinate, a feature different from that of ceNAC-1 (formerly known as ceNaDC1) and ceNAC-3 (formerly known as ceNaDC2). The transport process is electrogenic, as evidenced from the substrate-induced inward currents in oocytes expressing the transporter under voltage-clamp conditions. Expression studies using a reporter-gene fusion method in transgenic C. elegans show that the gene is expressed in the intestinal tract, the organ responsible for not only the digestion and absorption of nutrients but also for the storage of energy in this organism. Functional knockdown of the transporter by RNAi (RNA interference) not only leads to a significant increase in life span, but also causes a significant decrease in body size and fat content. The substrates of ceNAC-2 play a critical role in metabolic energy production and in the biosynthesis of cholesterol and fatty acids. The present studies suggest that the knockdown of these metabolic functions by RNAi is linked to an extension of life span and a decrease in fat content and body size.

Sodium-coupled and electrogenic transport of B-complex vitamin nicotinic acid by slc5a8, a member of the Na/glucose co-transporter gene family

SMCT (sodium-coupled monocarboxylate transporter; slc5a8) is a Na+-coupled transporter for lactate, pyruvate and short-chain fatty acids. Similar to these already known substrates of SMCT, the water-soluble B-complex vitamin nicotinic acid also exists as a monocarboxylate anion (nicotinate) under physiological conditions. Therefore we evaluated the ability of SMCT to mediate the uptake of nicotinate. In mammalian cells, the cloned mouse SMCT (slc5a8) induced the uptake of nicotinate. The SMCT-induced uptake was Na+-dependent. The Michaelis constant for the uptake process was 296+/-88 microM. The Na+-activation kinetics indicated that at least two Na+ ions are involved in the process. Among the various structural analogues tested, nicotinate was the most effective substrate. Nicotinamide and methylnicotinate were not recognized by the transporter. 2-pyrazine carboxylate and isonicotinate interacted with the transporter to a moderate extent. SMCT-mediated uptake of nicotinate was inhibited by lactate and pyruvate. In the Xenopus laevis oocyte expression system, SMCT-mediated nicotinate transport was electrogenic, as evident from the nicotinate-induced inward currents under voltage-clamp conditions. Substrate-induced currents in this expression system corroborated the substrate specificity determined in the mammalian cell expression system. The kinetic parameters with regard to the affinity of the transporter for nicotinate and the Hill coefficient for Na+ activation, determined by using the oocyte expression system, were also similar to those obtained from the mammalian cell expression system. We conclude that SMCT functions not only as a Na+-coupled transporter for short-chain fatty acids and lactate but also as a Na+-coupled transporter for the water-soluble vitamin nicotinic acid.

Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

Cell‐free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis‐based Escherichia coli cell‐free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light‐driven proton pump bacteriorhodopsin, consisting of seven transmembrane α‐helices. The cell‐free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3–0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent‐lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.

Comparison of the Hepatic Uptake Clearances of Fifteen Drugs with a Wide Range of Membrane Permeabilities in Isolated Rat Hepatocytes and Perfused Rat Livers

The hepatic uptake clearances of 15 ligands with a wide range of permeabilities were determined in rats using two techniques: centrifugal filtration with isolated hepatocytes and the multiple indicator dilution (MID) method with isolated perfused livers. Some of the uptake clearance values were taken from the literature. Uptake clearance values obtained from isolated hepatocytes were extrapolated to that per gram liver (PSinf, cell), assuming that 1 g of liver has 1.3 × 108 cells. The values of PSinf, cell varied from approximately 0.1 to 72 (mL/min/g liver). The values of PSinf, cell were similar to those (PSinf,MID) determined by the MID method for ligands with uptake clearances below approximately 1 mL/min/g liver. However, for the ligands with larger uptake clearances, the PSinf, MID values were lower than the PSinf,cell values and appeared to reach an upper limit (approx. 15–20 mL/min/g liver). The PSinf,cell values of 1-propranolol, tetraphenylphosphonium (TPP+), and diazepam were 72, 43, and 22 mL/min/g liver, respectively, whereas their uptake clearances (PSinf,MID) determined by the MID method were 4 to 10 times lower. One of the possible mechanisms for this discrepancy is that an unstirred water layer, which may exist in Disses space in isolated perfused livers (and probably under in vivo condition), limits the hepatic uptake rate of ligands with extremely high membrane permeabilities.

Functional features and genomic organization of mouse NaCT, a sodium-coupled transporter for tricarboxylic acid cycle intermediates

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.

Transport systems for opioid peptides in mammalian tissues.

Transmembrane transport of endogenous as well as synthetic opioid peptides is a critical determinant of pharmacokinetics and biologic efficacy of these peptides. This transport process influences the distribution of opioid peptides across the blood-brain barrier and their elimination from the body. A multitude of transport systems that recognize opioid peptides as substrates have been characterized at the functional level, and these transport systems are expressed differentially at different sites in the body. Many of these transport systems have been identified at the molecular level. These include the H+-coupled peptide transporters PEPT1 and PEPT2, the adenosine triphosphate-dependent efflux transporters P-glycoprotein and multidrug resistance-related protein 2, and several members of the organic anion-transporting polypeptide gene family. There are however many additional transport systems that are known to transport opioid peptides but their molecular identities still remain unknown.