Short-chain Fatty Acid Transport Research Articles

Transepithelial transport and intraepithelial metabolism of short-chain fatty acids (SCFA) in the porcine proximal colon are influenced by SCFA concentration and luminal pH

Short-chain fatty acids (SCFA) are end products of bacterial fermentation in the colon and cecum of monogastric animals. As SCFA serve as relevant energy suppliers for colonocytes and various tissues, it is important to reveal fundamental mechanistic characteristics of their transepithelial transport subjected to transient variations of fermentations rates. We performed Ussing chamber studies with porcine ( Sus scrofa) colon epithelium under physiological conditions and examined individual mucosal disappearance, metabolized loss, tissue concentrations and serosal release of acetate, propionate and butyrate by gas chromatography. Reduction of initial SCFA concentrations from 80 to 40 mmol/L resulted in diminished absolute flux rates, but the relative proportions of mucosal disappearance and intracellular metabolization of individual SCFA were slightly enhanced. Simulation of high fermentation rates by lowering the mucosal pH induced an increase in mucosal disappearance and serosal release of all SCFA, while their tissue contents trended to lower levels. With respect to the metabolization at lowered pH we found increased acetate concentrations and a decrease of propionate and butyrate. Our data indicate that the colon epithelium possesses a high adaptive capacity to ensure its energetic maintenance under various intraluminal fermentation rates by utilizing the unique features of individual SCFA as energy sources.

Abstract 2851: Genetic polymorphisms and expression of SLC5A8 in prostate cancer

Abstract Prostate cancer is the most common cancer in men, comprising approximately one-third of all male-specific cancers. SLC5A8 is a sodium-coupled transporter for short-chain fatty acids which are known to induce apoptosis by histone deacetylase inhibition. Previous studies suggest that SLC5A8 may function as a tumor suppressor gene whose silencing by epigenetic changes may contribute to carcinogenesis. For this reason, we investigated the role of expression and single nucleotide polymorphisms (SNPs) of SLC5A8 in prostate cancer risk and aggressiveness. We examined the association between common polymorphisms and risk for prostate cancer using a case-control study with 587 patients with incident primary prostate cancer, recruited at the Moffitt Cancer Center, and the James Haley VA Hospital and 347 age-matched controls, who were visiting the Lifetime Cancer Screening Center, which is affiliated with the Moffitt Cancer Center. All control subjects were male and had no previous diagnosis of cancer. We genotyped each study subject's DNA for four SNPs (rs164365, rs1709189, rs1399236, and rs1681096) in SLC5A8, by the TaqMan analysis. The frequencies of these SNPs were compared between cases and controls using logistic regression. Genotype distributions of all candidate SNPs in the controls followed Hardy-Weinberg equilibrium. In addition, SLC5A8 protein expression was estimated using innmunostained tissue microarray constructed from prostate tumor and normal tissues from BPH patients. We derived a semi-quantitative assessment by multiplying immunostain intensity and percentage of cells stained (range=0-9). We collected 140 prostate tumor tissues and 43 pairs of tumor and adjacent normal tissues from prostate cancer patients. From controls and BPH patients, 56 normal tissues were obtained. Among 43 pairs of tumor and normal tissues, SLC5A8 expression was significantly higher in tumor tissues than in their adjacent normal tissues (p<0.001). The similar result was shown in comparing tumor and normal tissues from independent individuals. SLC5A8 expression in 140 tumor tissues (median=4.5, IQR=3-6) was significantly higher than in 56 normal tissues (median=1.4, IQR=0-3, Wilcoxon test p<0.001). We observed a significant risk increase in individuals with at least one codon 490 SNP allele (rs164365) (OR=1.35, 95%=1.00-1.80) after adjusting for age and family history, especially among tall individuals (≥70 inches) (OR=1.83, 95%=1.22-2.73). Classification of patients by clinical aggressiveness did not show a significant difference of SLC5A8 genotype distribution and immunostating. Total 20 SNPs including 3 SNPs above in SLC5A8 were evaluated in the GWA study previously (CGEMS). Although these CGEMS data does not confirm the association between codon 490 polymorphism (rs164365) and prostate cancer risk, we observed significant SNP interactions associated with prostate cancer risk among individuals without a family history. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2851.

Role of Colonic Short-Chain Fatty Acid Transport in Diarrhea

Short-chain fatty acids (SCFA) are the major anion in stool and are synthesized from nonabsorbed carbohydrate by the colonic microbiota. Nonabsorbed carbohydrate are not absorbed in the colon and induce an osmotically mediated diarrhea; in contrast, SCFA are absorbed by colonic epithelial cells and stimulate Na-dependent fluid absorption via a cyclic AMP-independent process involving apical membrane Na-H, SCFA-HCO(3), and Cl-SCFA exchanges. SCFA production represents an adaptive process to conserve calories, fluid, and electrolytes. Inhibition of SCFA synthesis by antibiotics and administration of PEG, a substance that is not metabolized by colonic microbiota, both result in diarrhea. In contrast, increased production of SCFA as a result of providing starch that is relatively resistant to amylase digestion [so-called resistant starch (RS)] to oral rehydration solution (RS-ORS) improves the efficacy of ORS and represents an important approach to improve the effectiveness of ORS in the treatment of acute diarrhea in children under five years of age.

Rats consuming bran from black and brown sorghums have lower short chain fatty acid concentrations and fewer aberrant colonic crypts

Diets containing bran isolated from brown and black sorghum grain produce fewer (68 and 38%, respectively, P < 0.04) high multiplicity aberrant crypt foci (HMACF) in azoxymethane‐ (AOM) injected rats, relative to a cellulose diet. Because of the potential for short chain fatty acids (SCFA) to influence colon carcinogenesis, we determined SCFA concentrations in feces (reported as µM/g wet feces) from rats (n=10/diet) consuming diets containing 6% fiber from either cellulose or bran isolated from white, brown (contains tannins), or black (contains anthocyanins) sorghum. Diets were fed for 10 wk, with two AOM injections (15 mg/kg BW) given in wk 3 and 4. Total SCFA were affected (P < 0.0001) by diet; greatest for white sorghum bran (32.0 µM), intermediate for cellulose (22.6 µM), and lowest for black or brown sorghum bran (15.4 or 14.7 µM, respectively). Butyrate concentrations were affected (P < 0.0001) by diet; highest for white sorghum bran (7.8 µM), lowest for brown sorghum bran (1.07 µM), while concentrations were intermediate and similar for black sorghum bran and cellulose (2.7 and 3.6 µM, respectively). Differences among diets were the same when butyrate was expressed as a percentage of SCFA. These data suggest that there are significant changes either in the microbiota within the lumen or in the transport of SCFA into colonocytes, both of which can influence colon carcinogenesis. Funded by USDA 58‐5430‐5‐339 and NIEHS P30‐ES09106.

DNA hypermethylation and epigenetic silencing of the tumor suppressor gene, SLC5A8, in acute myeloid leukemia with the MLL partial tandem duplication

AbstractPosttranslationally modified histones and DNA hypermethylation frequently interplay to deregulate gene expression in cancer. We report that acute myeloid leukemia (AML) with an aberrant histone methyltransferase, the mixed lineage leukemia partial tandem duplication (MLL-PTD), exhibits increased global DNA methylation versus AML with MLL-wildtype (MLL-WT; P = .02). Among the differentially methylated genes, the SLC5A8 tumor suppressor gene (TSG) was more frequently hypermethylated (P = .003). In MLL-PTD+ cell lines having SLC5A8 promoter hypermethylation, incubation with decitabine activated SLC5A8 expression. Ectopic SLC5A8 expression enhanced histones H3 and H4 acetylation in response to the histone deacetylase inhibitor, valproate, consistent with the encoded protein—SMCT1—short-chain fatty acid transport function. In addition, enhanced cell death was observed in SMCT1-expressing MLL-PTD+ AML cells treated with valproate. Within the majority of MLL-PTD AML is a mechanism in which DNA hypermethylation silences a TSG that, together with MLL-PTD, can contribute further to aberrant chromatin remodeling and altered gene expression.

Sodium-Coupled Transport of the Short Chain Fatty Acid Butyrate by SLC5A8 and Its Relevance to Colon Cancer

IntroductionSLC5A8, expressed predominantly in the colon, is a Na+-coupled transporter for short-chain fatty acids. In this paper, we report on the characterization of butyrate transport by SLC5A8 and the relevance of SLC5A8-mediated butyrate transport to colon cancer. ResultsSLC5A8 transports butyrate via a Na+-dependent electrogenic process. Na+ activation of the transport process exhibits sigmoidal kinetics, indicating involvement of more than one Na+ in the activation process. SLC5A8 is silenced in colon cancer in humans, in a mouse model of intestinal/colon cancer, and in colon cancer cell lines. The tumor-associated silencing of SLC5A8 involves DNA methylation by DNA methyltransferase 1. Reexpression of SLC5A8 in colon cancer cells leads to apoptosis but only in the presence of butyrate. SLC5A8-mediated entry of butyrate into cancer cells is associated with inhibition of histone deacetylation. The changes in gene expression in SLC5A8/butyrate-induced apoptosis include upregulation of pro-apoptotic genes and downregulation of anti-apoptotic genes. In addition, the expression of phosphatidylinositol-3-kinase subunits is affected differentially, with downregulation of p85α and upregulation of p55α and p50α. ConclusionThese studies show that SLC5A8 mediates the tumor-suppressive effects of the bacterial fermentation product butyrate in the colon.

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

PURPOSE. To 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. We 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. [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 SMCTI. 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. Human SMCTI 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.

Expression of Monocarboxylate Transporter 1 (MCT1) in the Dog Intestine

In this study, the expression and distribution of monocarboxyolate transporter 1 (MCT1) along the intestines (duodenum, jejunum, ileum, cecum, colon and rectum) of dogs were investigated at both the mRNA and protein levels. The expression of MCT1 protein and its distribution were confirmed by Western blotting and immunohistochemical staining using the antibody for MCT1. We identified mRNA coding for MCT1 and a 43-kDa band of MCT1 protein in all regions from the duodenum to the rectum. Immunoreactive staining for MCT1 was also observed in epithelial cells throughout the intestines. MCT1 immunoreactivity was greater in the large intestine than in the small intestine. MCT1 protein was predominantly expressed on the basolateral membranes along intestinal epithelial cells, suggesting that MCT1 may play an important role in lactate efflux and transport of short-chain fatty acids (SCFAs) to the bloodstream across the basolateral membranes of the dog intestine.

Monocarboxylate transporter 1 (MCT1) plays a direct role in short‐chain fatty acids absorption in caprine rumen

Despite the importance of short-chain fatty acids (SCFA) in maintaining the ruminant physiology, the mechanism of SCFA absorption is still not fully studied. The goal of this study was to elucidate the possible involvement of monocarboxylate transporter 1 (MCT1) in the mechanism of SCFA transport in the caprine rumen, and to delineate the precise cellular localization and the level of MCT1 protein along the entire caprine gastrointestinal tract. RT-PCR revealed the presence of mRNA encoding for MCT1 in all regions of the caprine gastrointestinal tract. Quantitative Western blot analysis showed that the level of MCT1 protein was in the order of rumen >/= reticulum > omasum > caecum > proximal colon > distal colon > abomasum > small intestine. Immunohistochemistry and immunofluorescence confocal analyses revealed widespread immunoreactive positivities for MCT1 in the caprine stomach and large intestine. Amongst the stratified squamous epithelial cells of the forestomach, MCT1 was predominantly expressed on the cell boundaries of the stratum basale and stratum spinosum. Double-immunofluorescence confocal laser-scanning microscopy confirmed the co-localization of MCT1 with its ancillary protein, CD147 in the caprine gastrointestinal tract. In vivo and in vitro functional studies, under the influence of the MCT1 inhibitors, p-chloromercuribenzoate (pCMB) and p-chloromercuribenzoic acid (pCMBA), demonstrated significant inhibitory effect on acetate and propionate transport in the rumen. This study provides evidence, for the first time in ruminants, that MCT1 has a direct role in the transepithelial transport and efflux of the SCFA across the stratum spinosum and stratum basale of the forestomach toward the blood side.

Monocarboxylate transporter 1 (MCT1) mediates transport of short‐chain fatty acids in bovine caecum

The present study was undertaken to investigate the functional role of monocarboxylate transporter 1 (MCT1) in the ruminant large intestine. Messenger RNA encoding for MCT1 was verified by reverse transcriptase-polymerase chain reaction in caecum, proximal colon and distal colon of adult cattle. Both immunohistochemistry and confocal laser microscopy verified that the MCT1 protein was abundant in the surface epithelium of the large intestine, and the amount decreased from the opening of the crypt to its base. In the immunopositive cells, MCT1 was primarily localized in the basolateral membranes of epithelium lining the large intestine. Western blotting indicated that the levels of MCT1 protein were highest in the caecum, followed by proximal colon and then distal colon. In vitro studies were conducted to elucidate the possible involvement of MCT1 in the transport of short-chain fatty acids (SCFA) across the isolated mucosal sheets of cattle caecum using the Ussing chamber technique. Acetate absorption was found to be pH dependent, and the rate of acetate absorption increased as pH decreased. The serosal application of the MCT1 inhibitor 'p-chloromercuribenzoic acid (pCMB)' significantly reduced the transport of acetate across the caecal epithelium of cows. In addition, the transport of acetate was significantly reduced in the presence of its analogue, propionate, indicating that acetate and propionate compete for binding to the same transporter. The results show that MCT1 is a major route for SCFA efflux across the basolateral membrane of bovine large intestine and that it could play a role in the regulation of intracellular pH.

Expression of CD147 and monocarboxylate transporters MCT1, MCT2 and MCT4 in porcine small intestine and colon

Lactate, formed mainly in the stomach and small intestines, and short-chain fatty acids (SCFAs) formed in the colon, are ionised and require transporter proteins such as monocarboxylate transporters (MCTs) for absorption. The amounts of MCT1, MCT2, MCT4 and CD147, an ancillary protein for MCT1 and MCT4, were measured by immunoblotting the small intestine and colon of 40 pigs (Landrace, Yorkshire and Landrace × Yorkshire). MCT1 and MCT4 were found in both small intestine and colon, but MCT2 only in the small intestine. In both small intestine and colon, Yorkshire pigs had more CD147 than Landrace pigs, while no interbreed differences were found in MCT isoforms. Since CD147 is essential for the activity of MCT1 and MCT4, the breed difference suggests that MCT activity is higher in Yorkshire than in Landrace pigs. The absence of MCT2 in the colon suggests that it is mainly a lactate transporter, while MCT1 and MCT4 facilitate the transport of both lactate and SCFA.

Identity of SMCT1 (SLC5A8) as a neuron‐specific Na+‐coupled transporter for active uptake ofl‐lactate and ketone bodies in the brain

SMCT1 is a sodium-coupled (Na(+)-coupled) transporter for l-lactate and short-chain fatty acids. Here, we show that the ketone bodies, beta-d-hydroxybutyrate and acetoacetate, and the branched-chain ketoacid, alpha-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 beta-d-hydroxybutyrate, 0.21 +/- 0.04 mm for acetoacetate and 0.21 +/- 0.03 mm for alpha-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. SMCT1 protein 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.

Serrated Pathway and APC (Conventional)-Type Colorectal Polyps

This review addresses the genetic mutations and cell signaling pathway alterations in colorectal premalignant polyps, focusing on the link between molecular changes and morphologic features. Biallelic APC (adenomatous polyposis coli) mutations are directly responsible for the specific and characteristic cytologic features of dysplastic cells in conventional tubular adenomas. Sessile serrated adenomas (SSAs) are the precursor lesions of the serrated neoplasia pathway. The BRAF activating mutation and hypermethylation of SLC5A8, which mediates short chain fatty acid transport, may be the important events in the genesis of SSAs. Intracellular butyrate inhibits histone deacetylase, allowing histone hyperacetylation and, eventually, transcriptional activation of specific genes. Decreased p21(WAF1/CIP1) and activation of the mitogen-activated protein kinase pathway may be the key intermediary alterations. Progressive loss of cell cycle control and decreased and altered cytoplasmic differentiation produce the characteristic constellation of morphologic changes of SSAs and traditional serrated adenomas.

The Human Monocarboxylate Transporter MCT1: Gene Structure and Regulation

To the Editor: We read with concern the recent paper by Hadjiagapiou et al. ([4][1]) that examines the regulation of the monocarboxylate transporter isoform 1 (MCT1) gene promoter in Caco-2 cells and would like to make a number of comments with regard to 1 ) errors in their data and 2 ) misquotation

Expression and membrane localization of MCT isoforms along the length of the human intestine

Recent studies from our laboratory and others have demonstrated the involvement of monocarboxylate transporter (MCT)1 in the luminal uptake of short-chain fatty acids (SCFAs) in the human intestine. Functional studies from our laboratory previously demonstrated kinetically distinct SCFA transporters on the apical and basolateral membranes of human colonocytes. Although apical SCFA uptake is mediated by the MCT1 isoform, the molecular identity of the basolateral membrane SCFA transporter(s) and whether this transporter is encoded by another MCT isoform is not known. The present studies were designed to assess the expression and membrane localization of different MCT isoforms in human small intestine and colon. Immunoblotting was performed with the purified apical and basolateral membranes from human intestinal mucosa obtained from organ donor intestine. Immunohistochemistry studies were done on paraffin-embedded sections of human colonic biopsy samples. Immunoblotting studies detected a protein band of approximately 39 kDa for MCT1, predominantly in the apical membranes. The relative abundance of MCT1 mRNA and protein increased along the length of the human intestine. MCT4 (54 kDa) and MCT5 (54 kDa) isoforms showed basolateral localization and were highly expressed in the distal colon. Immunohistochemical studies confirmed that human MCT1 antibody labeling was confined to the apical membranes, whereas MCT5 antibody staining was restricted to the basolateral membranes of the colonocytes. We speculate that distinct MCT isoforms may be involved in SCFA transport across the apical or basolateral membranes in polarized colonic epithelial cells.

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.

Role of short-chain fatty acids in colonic HCO3secretion

Luminal isobutyrate, a relatively poor metabolized short-chain fatty acid (SCFA), induces HCO(3) secretion via a Cl-independent, DIDS-insensitive, carrier-mediated process as well as inhibiting both Cl-dependent and cAMP-induced HCO(3) secretion. The mechanism(s) responsible for these processes have not been well characterized. HCO(3) secretion was measured in isolated colonic mucosa mounted in Lucite chambers using pH stat technique and during microperfusion of isolated colonic crypts. (14)C-labeled butyrate, (14)C-labeled isobutyrate, and (36)Cl uptake were also determined by apical membrane vesicles (AMV) isolated from surface and/or crypt cells. Butyrate stimulation of Cl-independent, DIDS-insensitive 5-nitro-3-(3-phenylpropyl-amino)benzoic acid-insensitive HCO(3) secretion is greater than that by isobutyrate, suggesting that both SCFA transport and metabolism are critical for HCO(3) secretion. Both lumen and serosal 25 mM butyrate inhibit cAMP-induced HCO(3) secretion to a comparable degree (98 vs. 90%). In contrast, Cl-dependent HCO(3) secretion is downregulated by lumen 25 mM butyrate considerably more than by serosal butyrate (98 vs. 37%). Butyrate did not induce HCO(3) secretion in isolated microperfused crypts, whereas an outward-directed HCO(3) gradient-driven induced (14)C-butyrate uptake by surface but not crypt cell AMV. Both (36)Cl/HCO(3) exchange and potential-dependent (36)Cl movement in AMV were inhibited by 96-98% by 20 mM butyrate. We conclude that 1) SCFA-dependent HCO(3) secretion is the result of SCFA transport across the apical membrane via a SCFA/HCO(3) exchange more than intracellular SCFA metabolism; 2) SCFA-dependent HCO(3) secretion is most likely a result of an apical membrane SCFA/HCO(3) exchange in surface epithelial cells; 3) SCFA downregulates Cl-dependent and cAMP-induced HCO(3) secretion secondary to SCFA inhibition of apical membrane Cl/HCO(3) exchange and anion channel activity, respectively.

Histochemical demonstration of a Na+-coupled transporter for short-chain fatty acids (Slc5a8) in the intestine and kidney of the mouse

Short-chain fatty acids in the intestinal lumen affect colonic cell proliferation as well as function as an energy source for intestinal epithelial cells. A novel transporter of monocarboxylates, Slc5a8, is expressed abundantly in the colon, where it may participate in the Na(+)-coupled absorption of short-chain fatty acids produced by bacterial fermentation of dietary fiber. The present study examined the cellular localization of Slc5a8 in the murine gastrointestinal tract and kidney by in situ hybridization and immunohistochemistry. The hybridization signals were recognized in the terminal ileum and whole length of the large intestine, and were especially intense in the distal colon and rectum. The immunoreactivity of Slc5a8 was restricted to the striated border (the brush border) of enterocytes, and was not present in goblet cells, Paneth cells, or lamina propria cells. In the kidney, proximal tubules of both the cortex and the outer stripe of the outer medulla intensely expressed Slc5a8 mRNA, while the distal portions, including the loop of Henle, lacked the signals. The renal Slc5a8 immunoreactivity was localized only in the brush border of proximal tubules, not along the basolateral membrane. Thyroid follicular cells were immunoreactive for Slc5a8, with predominant labeling on the apical membrane. No other organs, including the esophagus, stomach, liver, pancreas, and salivary glands contained any notable signals of Slc5a8. These findings on the cellular and subcellular localization of Slc5a8 under normal conditions are helpful for understanding the physiological and pathological roles of Slc5a8.

Expression of slc5a8 in Kidney and Its Role in Na+-coupled Transport of Lactate

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.

Functional Identification of SLC5A8, a Tumor Suppressor Down-regulated in Colon Cancer, as a Na+-coupled Transporter for Short-chain Fatty Acids

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 voltage-clamp 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.