Lactaturia and Loss of Sodium-dependent Lactate Uptake in the Colon of SLC5A8-deficient Mice
2008; Elsevier BV; Volume: 283; Issue: 36 Linguagem: Inglês
10.1074/jbc.m802681200
ISSN1083-351X
AutoresHenning Frank, Nicole Gröger, Martin Diener, Christoph Becker, Thomas Braun, Thomas Boettger,
Tópico(s)Biochemical Analysis and Sensing Techniques
ResumoSLC5A8 is a member of the sodium/glucose cotransporter family. It has been proposed that SLC5A8 might act as an apical iodide transporter in the thyroid follicular cells or as a transporter of short chain monocarboxylates. We have directly addressed the functional role of SLC5A8 in vivo by generation of SLC5A8 mutant mice. We found that SLC5A8 is responsible for the re-absorption of lactate at the apical membrane of the kidney proximal tubules and of serous salivary gland ducts. In addition, SLC5A8 mediated the uptake of lactate into colonocytes under physiological conditions. We did not find any evidence of SLC5A8 being essential for the apical iodide transport in the thyroid gland, even if the ion-cotransporter SLC26A4, causing the human Pendred syndrome, is missing. Because SLC5A8 is transcriptionally silenced in many tumors, it has been proposed that SLC5A8-mediated transport of butyrate suppresses tumor formation. Treatment of Slc5a8-/- mice with carcinogens and breeding to the Apcmin mouse line did not reveal a higher incidence of tumor formation. We conclude that SLC5A8 is instrumental in preventing lactaturia and loss of sodium-dependent uptake of lactate in the colon but does not have any apparent role in the prevention of tumor formation and growth. SLC5A8 is a member of the sodium/glucose cotransporter family. It has been proposed that SLC5A8 might act as an apical iodide transporter in the thyroid follicular cells or as a transporter of short chain monocarboxylates. We have directly addressed the functional role of SLC5A8 in vivo by generation of SLC5A8 mutant mice. We found that SLC5A8 is responsible for the re-absorption of lactate at the apical membrane of the kidney proximal tubules and of serous salivary gland ducts. In addition, SLC5A8 mediated the uptake of lactate into colonocytes under physiological conditions. We did not find any evidence of SLC5A8 being essential for the apical iodide transport in the thyroid gland, even if the ion-cotransporter SLC26A4, causing the human Pendred syndrome, is missing. Because SLC5A8 is transcriptionally silenced in many tumors, it has been proposed that SLC5A8-mediated transport of butyrate suppresses tumor formation. Treatment of Slc5a8-/- mice with carcinogens and breeding to the Apcmin mouse line did not reveal a higher incidence of tumor formation. We conclude that SLC5A8 is instrumental in preventing lactaturia and loss of sodium-dependent uptake of lactate in the colon but does not have any apparent role in the prevention of tumor formation and growth. Slc5a8 belongs to the sodium/glucose cotransporter family Slc5. Loss of function of these transporters may cause intestinal glucose/galactose re-absorption defects, glucosurea, or in the case of the Na+/I- symporter (NIS, 2The abbreviations used are:NISNa+/I- symporterWTwild typeKOknock-outSMCTsodium monocarboxylate transporterTSHthyroid-stimulating hormoneTRITCtetramethylrhodamine isothiocyanate. SLC5A5) hypothyroidism in humans (1Wright E.M. Turk E. Pflugers Arch. 2004; 447: 510-518Crossref PubMed Scopus (27) Google Scholar). NIS is expressed in the basolateral membrane of thyroid follicular cells and mediates the uptake of iodide from blood into the cytoplasm of thyrocytes. Iodide is transported across the cytoplasm to the apical membrane toward the follicle lumen where it is oxidized and bound to thyroglobulin. A candidate molecule to mediate the efflux of iodide at the apical membrane is SLC26A4, which is mutated in Pendred syndrome, a genetic disorder that is associated with profound sensorineural hearing loss and goiter (2Fraser G.R. Ann. Hum. Genet. 1965; 28: 201-249Crossref PubMed Scopus (204) Google Scholar, 3Everett L.A. Glaser B. Beck J.C. Idol J.R. Buchs A. Heyman M. Adawi F. Hazani E. Nassir E. Baxevanis A.D. Sheffield V.C. Green E.D. Nat. Genet. 1997; 17: 411-422Crossref PubMed Scopus (1004) Google Scholar). It has been shown in vitro in a polarized cell system that SLC26A4 can mediate the efflux of iodide via the apical membrane of a cell (4Gillam M.P. Sidhaye A.R. Lee E.J. Rutishauser J. Stephan C.W. Kopp P. J. Biol. Chem. 2004; 279: 13004-13010Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Nevertheless, a knockout mouse model for Slc26a4 does not show any signs of thyroid dysfunction, challenging the role of Pendrin in iodide transport at the apical membrane of thyrocytes (5Everett L.A. Belyantseva I.A. Noben-Trauth K. Cantos R. Chen A. Thakkar S.I. Hoogstraten-Miller S.L. Kachar B. Wu D.K. Green E.D. Hum. Mol. Genet. 2001; 10: 153-161Crossref PubMed Scopus (348) Google Scholar). SLC5A8 was first described as a close structural relative of human NIS (46% identity and 70% similarity), but in contrast to NIS, it is expressed at the apical membrane of thyrocytes (6Rodriguez A.M. Perron B. Lacroix L. Caillou B. Leblanc G. Schlumberger M. Bidart J.M. Pourcher T. J. Clin. Endocrinol. Metab. 2002; 87: 3500-3503Crossref PubMed Scopus (119) Google Scholar). The expression of SLC5A8 reduces iodide accumulation in NIS-transfected COS-7 cells, compatible with a role of SLC5A8 as a transporter that may mediate the flux of iodide ions into the follicular lumen of the thyroid (6Rodriguez A.M. Perron B. Lacroix L. Caillou B. Leblanc G. Schlumberger M. Bidart J.M. Pourcher T. J. Clin. Endocrinol. Metab. 2002; 87: 3500-3503Crossref PubMed Scopus (119) Google Scholar). Thus, SLC5A8 was designated human Apical Iodide Transporter (hAIT). Later it was shown that SLC5A8 overexpressed in Xenopus laevis oocytes mediates the sodium-coupled transport of monocarboxylates like l-lactate, propionate, butyrate, or nicotinate, whereas exposure to iodide or bromate did not lead to currents (7Coady M.J. Chang M.H. Charron F.M. Plata C. Wallendorff B. Sah J.F. Markowitz S.D. Romero M.F. Lapointe J.Y. J. Physiol. 2004; 557: 719-731Crossref PubMed Scopus (139) Google Scholar, 8Miyauchi S. Gopal E. Fei Y.J. Ganapathy V. J. Biol. Chem. 2004; 279: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 9Paroder V. Spencer S.R. Paroder M. Arango D. Schwartz Jr., S. Mariadason J.M. Augenlicht L.H. Eskandari S. Carrasco N. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7270-7275Crossref PubMed Scopus (94) Google Scholar). The sodium-dependent transport of monocarboxylates by SLC5A8 was also corroborated in a mammalian expression system (10Gopal E. Fei Y.J. Sugawara M. Miyauchi S. Zhuang L. Martin P. Smith S.B. Prasad P.D. Ganapathy V. J. Biol. Chem. 2004; 279: 44522-44532Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) leading to renaming of the molecule as Sodium Monocarboxylate Transporter 1 (SMCT 1). Na+/I- symporter wild type knock-out sodium monocarboxylate transporter thyroid-stimulating hormone tetramethylrhodamine isothiocyanate. Because of glomerular filtration of lactate from blood to primary urine, lactate needs to be re-absorbed efficiently in the kidney to prevent lactaturia. Several studies suggest a sodium-dependent lactate re-absorption in the brush border membrane of the renal proximal tubule (11Barac-Nieto M. Murer H. Kinne R. Am. J. Physiol. 1980; 239: F496-F506PubMed Google Scholar). SLC5A8 has been considered to be instrumental in lactate re-absorption in the S3 segment of the renal proximal tubule (10Gopal E. Fei Y.J. Sugawara M. Miyauchi S. Zhuang L. Martin P. Smith S.B. Prasad P.D. Ganapathy V. J. Biol. Chem. 2004; 279: 44522-44532Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 12Thangaraju M. Ananth S. Martin P.M. Roon P. Smith S.B. Sterneck E. Prasad P.D. Ganapathy V. J. Biol. Chem. 2006; 281: 26769-26773Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) together with the related transporter SLC5A12 (SMCT2) in the S1 and S2 segment of the proximal tubule (12Thangaraju M. Ananth S. Martin P.M. Roon P. Smith S.B. Sterneck E. Prasad P.D. Ganapathy V. J. Biol. Chem. 2006; 281: 26769-26773Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 13Srinivas S.R. Gopal E. Zhuang L. Itagaki S. Martin P.M. Fei Y.J. Ganapathy V. Prasad P.D. Biochem. J. 2005; 392: 655-664Crossref PubMed Scopus (105) Google Scholar, 14Gopal E. Umapathy N.S. Martin P.M. Ananth S. Gnana-Prakasam J.P. Becker H. Wagner C.A. Ganapathy V. Prasad P.D. Biochim. Biophys. Acta. 2007; 1768: 2690-2697Crossref PubMed Scopus (69) Google Scholar). However, clear evidence for the role of SLC5A8 in lactate re-absorption in the kidney is still lacking. In addition to its physiological function in homeostasis of short monocarboxylates, a number of reports suggested a role for SLC5A8 in carcinogenesis. Because exon 1 of the SLC5A8 gene is aberrantly methylated in 59% of primary human colon cancers and in 52% of the analyzed colon cancer cell lines, it was proposed that SLC5A8 might act as a tumor suppressor gene (15Li H. Myeroff L. Smiraglia D. Romero M.F. Pretlow T.P. Kasturi L. Lutterbaugh J. Rerko R.M. Casey G. Issa J.P. Willis J. Willson J.K. Plass C. Markowitz S.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8412-8417Crossref PubMed Scopus (250) Google Scholar). Similarly, re-expression of SLC5A8 in colon cancer cell lines lacking SLC5A8 suppressed colony growth (15Li H. Myeroff L. Smiraglia D. Romero M.F. Pretlow T.P. Kasturi L. Lutterbaugh J. Rerko R.M. Casey G. Issa J.P. Willis J. Willson J.K. Plass C. Markowitz S.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8412-8417Crossref PubMed Scopus (250) Google Scholar). Potential tumor suppressive effects of SLC5A8 were also demonstrated for colon cancer (9Paroder V. Spencer S.R. Paroder M. Arango D. Schwartz Jr., S. Mariadason J.M. Augenlicht L.H. Eskandari S. Carrasco N. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7270-7275Crossref PubMed Scopus (94) Google Scholar), gastric cancer (16Ueno M. Toyota M. Akino K. Suzuki H. Kusano M. Satoh A. Mita H. Sasaki Y. Nojima M. Yanagihara K. Hinoda Y. Tokino T. Imai K. Tumour Biol. 2004; 25: 134-140Crossref PubMed Scopus (59) Google Scholar), thyroid cancer (17Porra 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. Rousset B. J. Clin. Endocrinol. Metab. 2005; 90: 3028-3035Crossref PubMed Scopus (95) Google Scholar), and oligodendroglioma (18Hong C. Maunakea A. Jun P. Bollen A.W. Hodgson J.G. Goldenberg D.D. Weiss W.A. Costello J.F. Cancer Res. 2005; 65: 3617-3623Crossref PubMed Scopus (58) Google Scholar). It was claimed that SLC5A8-mediated transport of butyrate might serve as the molecular basis for the tumor suppressive role of SLC5A8 (19Ganapathy V. Gopal E. Miyauchi S. Prasad P.D. Biochem. Soc. Trans. 2005; 33: 237-240Crossref PubMed Scopus (85) Google Scholar). Here we demonstrate that SLC5A8 is expressed in the apical membranes of cells in the kidney, intestine, thyroid, and salivary gland, but not in the brain, as defined by RNA in situ hybridization and a newly derived antibody directed against the C terminus of the protein. The generation of a loss of function mouse model of Slc5a8 revealed that SLC5A8 is essential for lactate re-absorption in the kidney and for adjusting lactate concentration in saliva. But it is dispensable for iodide transport in the thyroid gland even in the absence of the ion-cotransporter SLC26A4 responsible for the human Pendred syndrome. In the colon, we detected a sodium-dependent current induced by an addition of lactate to the luminal side that was mediated by SLC5A8, while no evidence was found for a SLC5A8-dependent butyrate transport in the colon. Our study reveals that SLC5A8 has no apparent role in the prevention of tumor formation and growth in the colon. Targeting Construct and Mutant Mice—The mouse genomic clone was derived from a 129/ola cosmid library. A loxP-flanked neomycin-resistance cassette was inserted into a BamHI site in front of the fourth exon. A loxP site was inserted into the HpaI site following the fifth exon of Slc5a8 (NP_145423.2). The resulting targeting vector contained 10.9 kb of 5′- and 6.1 kb of 3′-flanking sequence (Fig. 1A). Mouse embryonic stem cells were electroporated, and mouse lines recombinant for the Slc5a8 locus were established from two independent ES clones. Heterozygous animals of each clone were mated to Meu-Cre40 mice (20Leneuve P. Colnot S. Hamard G. Francis F. Niwa-Kawakita M. Giovannini M. Holzenberger M. Nucleic Acids Res. 2003; 31: e21Crossref PubMed Scopus (52) Google Scholar) to remove the neoR-cassette and 2.7 kb genomic sequence. The loss of exons 4 and 5 results in a frameshift and truncation of the SLC5A8 protein after 4 of at least 10 transmembrane domains. The study was performed in a mixed 129SV/C57Bl6 background. ES cells and mice were genotyped using a BamHI digest and a 3′ probe (Fig. 1H). The ApcMin strain is commercially available from Jackson Laboratory. The Slc26a4 knock-out mice were provided as cryoconserved embryos by Eric Green (5Everett L.A. Belyantseva I.A. Noben-Trauth K. Cantos R. Chen A. Thakkar S.I. Hoogstraten-Miller S.L. Kachar B. Wu D.K. Green E.D. Hum. Mol. Genet. 2001; 10: 153-161Crossref PubMed Scopus (348) Google Scholar). In Situ Hybridization, Immunofluorescence, and Western Blot—For in situ hybridization, 12-μm cryosections were prepared. The templates for probe transcription were 1175 bp of Slc5a8 cDNA (5′ cgtacacatgatgcttcagttttgg, 3′ catgaaacaaaccacatgacgtgtg) and of 994 bp of Slc5a12 cDNA (5′ tgtactttaacttgttgggtctctgg, 3′ gggttgtagcctggaatctgagccaa) amplified by RT-PCR. A polyclonal antiserum was raised against a peptide sequence (VFKKRNHVLNYKLHPVEVGC) close to the SLC5A8 C terminus. The serum was affinity-purified against the peptide sequence. Other primary antibodies were α-MCT 1 (1:300; Chemicon), α-laminin (1:100; DSHB), α-fibronectin (1:100; BD Biosciences); Secondary antibodies (1:2000) were Alexa Fluor 488 goat α-mouse, Alexa Fluor 546 goat α-rabbit, Alexa Fluor 488 goat α-rabbit (Invitrogen), or sheep α-rabbit IgG-HRP (Chemicon, 1:5000). TOTO-3 or DAPI (both 1:1000; Invitrogen) were used for nuclear staining, Phalloidin-TRITC (1:1000, Sigma) for actin labeling. For immunofluorescence, appropriate tissues of Slc5a8-/- mice were used as controls. Protein extracts from tissues were prepared by homogenization in 3 ml of buffer (125 mm NaCl, 20 mm Tris/HCl, pH 8.0, 4.5 mm EDTA, Complete protease inhibitor mixture (Roche 11836153001). Extracts were centrifuged at 1000 × g for 10 min. From the supernatant, proteins were collected at 135,000 × g for 1 h. The pellet was resuspended in 100 μl of sample buffer (100 mm Tris/HCl, pH 8.0, 10 mm EDTA, 40 mm dithiothreitol, 10% SDS, Complete protease inhibitor mixture). The protein concentration was determined using the DC Protein Assay kit (Bio-Rad 500-0111). 10 μg of protein extracts (0.3 μg for kidney) were separated on 4–12% SDS-PAGE gradient gels and transferred onto nitrocellulose membranes (Invitrogen). By using horseradish peroxidase-coupled secondary antibody and SuperSignal Femto detection solution (Perbio Science), signals were detected with a VersaDoc Imaging System (Bio-Rad). Metabolic Cages—Single mice were kept in mouse metabolic cages (Techniplast) with free access to water and chow. After adaptation to the cage, urine and feces were collected over 24-h periods. The analysis of urinary electrolytes was performed by Vet Med Lab (Ludwigsburg, Germany). Measurement of Lactate in Blood and Saliva—Blood for analysis of lactate concentration was collected from the tail tip. For saliva collection, animals were fasted for 5 h before the experiment. To activate salivation, mice were injected with 5 mg of pilocarpine per kg of bodyweight. After 2 and 4 min, saliva was collected from the mouth of the animals. The lactate concentration was determined by Lactate Scout (SensLab, Germany). Histology—Tissue samples (large intestine, kidney, submandibular gland, and thyroid) were perfused and fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. Samples were stained with hematoxylin and eosin and examined for histological changes by light microscopy. Carcinogen Treatment—Treatments were performed with 5-month-old mice. A single lot of azoxymethane (AOM) was used (Sigma-Aldrich, 100-mg isovials). Each 100-mg vial was resuspended in 3.9 ml of phosphate-buffered saline and aliquots were stored at -20 °C. A 1 μg/μl working solution was prepared in 0.9% NaCl. Mice were treated intraperitoneally with 10 mg of AOM per kg of body weight once a week for 8 weeks (21Bissahoyo A. Pearsall R.S. Hanlon K. Amann V. Hicks D. Godfrey V.L. Threadgill D.W. Toxicol. Sci. 2005; 88: 340-345Crossref PubMed Scopus (93) Google Scholar, 22Nambiar P.R. Girnun G. Lillo N.A. Guda K. Whiteley H.E. Rosenberg D.W. Int. J. Oncol. 2003; 22: 145-150PubMed Google Scholar). Second, 2% sodium dextran sulfate (MW: 36,000–50,000, MP Biomedicals, Inc.) was added to the drinking water for 1 week, followed by a 2-week-period with drinking water. This treatment was repeated three times (23Okayasu I. Hatakeyama S. Yamada M. Ohkusa T. Inagaki Y. Nakaya R. Gastroenterology. 1990; 98: 694-702Abstract Full Text PDF PubMed Google Scholar). Three months after the end of the respective treatment, tumor development was scored by endoscopic examination as previously described (24Becker C. Fantini M.C. Neurath M.F. Nat. Protoc. 2006; 1: 2900-2904Crossref PubMed Scopus (249) Google Scholar). In brief, all visible tumors were counted and graded by the following criteria: grade 1-very small but detectable; grades 2–5: tumor covering up to one-eighth [2], up to one-fourth [3] up to one-half [4], more than one-half [5] of the colonic circumference. RNA Expression Analysis—Total RNA from colon was isolated with the TRIzol (Invitrogen) method. RNA concentrations were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). RNA quality was confirmed using RNA 6000 Nano LabChip kit (Agilent 2100 Bioanalyzer). RNA integrity numbers for the 10 samples ranged from 8.9 to 9.6. For Affymetrix GeneChip Mouse Genome 430 2.0 hybridization, 10 μg of total RNA were used for sample preparations as suggested by the Affymetrix Eukaryotic Target Protocol. Affymetrix Quality Control Reporter tool was used to assure the uniform performance of the individual GeneChip hybridizations with present calls between 52 and 56% for all GeneChips and comparable 3′/5′ ratios for the internal controls. Data were analyzed using Stratagene Arrayassist 5.5.1. For background correction, normalization, and probe summarization we used the RMA algorithm (25Irizarry R.A. Hobbs B. Collin F. Beazer-Barclay Y.D. Antonellis K.J. Scherf U. Speed T.P. Biostatistics. 2003; 4: 249-264Crossref PubMed Scopus (8483) Google Scholar). To suppress noise at very low signal intensities, variance stabilization was performed. Data were log-transformed to base 2 and baseline transformed with the WT group as baseline. An asymptotic t test was performed. To account for multiple hypothesis testing, Benjamini-Hochberg (26Benjamini Y. Hochberg Y. J. R. Statist. Soc. 1995; 57: 289-300Google Scholar) and alternatively Westfall-Young (27Westfall P.H. Young S.S. Resampling-based Multiple Testing: Examples and Methods for p-Value Adjustment. John Wiley and Sons, New York1993Google Scholar) correction were performed. For real-time RT-PCR we used an iCycler iQ™ (Bio-Rad) using the Absolute QPCR SYBR Green Fluorescein mix (Abgene). Values were normalized to an endogenous reference: SQtarget/SQHPRT. Analysis of Thyroid Function—The T4 concentration was measured by using a commercial RIA (Diagnostic Systems Laboratories, Inc., Beckmann Coulter). As an internal control, we used serum from mice fed with iodide-depleted nutrition (Altromin) containing 0.15% propylthiouracil (PTU, Sigma) for 10 days. TSH measurement was performed by Dr. Parlow (National Hormone & Peptide Program; Harbor-UCLA). The efficiency of iodide incorporation into thyroglobulin was measured as described (28van den Hove M.F. Croizet-Berger K. Jouret F. Guggino S.E. Guggino W.B. Devuyst O. Courtoy P.J. Endocrinology. 2006; 147: 1287-1296Crossref PubMed Scopus (72) Google Scholar). Briefly, we injected 15 μCi of 125I- (Amersham Biosciences) intraperitoneally. After 1 h, animals were anesthetized; thereafter blood was taken from the heart with a syringe and the thyroid was excised. To block unspecific binding of 125I to hemoglobin, iodide to a final concentration of 6 mm was added to the blood sample (29Harmatz P.R. Walsh M.K. Walker W.A. Hanson D.G. Bloch K.J. J. Immunol. Methods. 1987; 102: 213-219Crossref PubMed Scopus (4) Google Scholar). Unbound 125I- was removed from serum by Zebra™ Desalt Spin Columns (Pierce). The ratio protein-bound 125I/free serum 125I- over time is a measure of thyroid function. A second readout for 125I- organification was the ratio protein-bound 125I in serum/125I- taken up into the thyroid over a 1-h period. To evaluate the method, a time course series of the iodide incorporation was performed. A 1-h period was chosen between 125I- injection and blood sampling. Ussing Chamber Analysis—A 3-cm long proxomedial part of the colon was removed, and the colonic lumen was rinsed with ice-cold Parsons solution. This standard buffer for the Ussing chamber experiments contained (mm): 107 NaCl, 4.5 KCl, 25 NaHCO3, 1,8 Na2HPO4, 0.2 NaH2PO4, 1.25 CaCl2, 1 MgSO4, and 12 glucose. The solution was gassed with carbogen; pH was 7.4. The addition of lactate did not change this pH. For the Na+-free solution, NaCl was replaced by N-methyl-d-glucamine (NMDG+) chloride. The tissue was fixed in a modified Ussing chamber, bathed with a volume of 3.5 ml on each side of the mucosa, and short-circuited by a computer-controlled voltage-clamp device (Muβler Ingenieurbüro, Aachen, FRG) with a correction for solution resistance. The exposed surface of the tissue was 1 cm2. Short-circuit current (Isc) was continuously recorded, and tissue conductance (Gt) was measured every min by applying a current pulse of ± 50 μA·cm-2. Isc is expressed as μEq·h-1·cm-2, i.e. the flux of a monovalent ion per time and area, with 1 μEq·h-1·cm-2 = 26.9 μA·cm-2. For statistical analysis, we calculated the ratio between the maximal decrease of the Isc within 5 min after lactate addition and the mean Isc 3 min before lactate addition. To determine the unidirectional mucosa-to-serosa flux (Jms) of butyrate, sodium [14C]butyrate (37 kBq, Hartmann Analytical, Braunschweig, Germany) was added to the mucosal side after an equilibration period of 30 min. For these experiments, the mucosal buffer contained 5 mm sodium butyrate and the serosal buffer contained 8 mm mannitol for osmotic compensation. After an additional 30 min to allow isotope fluxes to reach a steady state, unidirectional ion fluxes were determined over two sequential 20-min periods. Both periods were averaged to calculate the flux rate. All aliquots from the labeled side were replaced by unlabeled buffer solution, and an appropriate correction for this replacement solution was performed. The radioactivity in the samples was determined in a liquid scintillation counter (TRIS-CARB® 2700 TR Liquid scintillation analyser, Packard, Frankfurt, Germany). The unidirectional mucosa-to-serosa flux (Jms) is indicated as transported amount of butyrate per surface area and time [μmol·cm-2·h-1]. Statistics—An unpaired Student's t test assuming equal variances or a Mann-Whitney test was used to compare data from two treatment groups. Homozygous Mutant SLC5A8 Mice Are Viable and Fertile and Lack Major Morphological Malformations—The Slc5a8 locus was modified in ES cells by targeted recombination resulting in the integration of a neomycin-resistance cassette flanked by loxP sites upstream of the fourth exon and an additional loxP site downstream of the fifth exon (Fig. 1A). Mice homozygous for the integrated targeting vector did not show any obvious phenotype. To delete the selection cassette and generate a null allele of Slc5a8, mutant mice were bred to Meu-Cre40 mice resulting in the deletion of a 2.7-kb fragment encompassing the fourth and fifth exon (Fig. 1A). Heterozygous progeny, which were Cre-recombinase negative were backcrossed to C57BL6 mice and mated to each other to generate homozygous mutant animals. Homozygous animals were viable and fertile and did not show any obvious phenotype regarding weight, morphology, or motility. SLC5A8 Is Expressed in Apical Membranes of Different Epithelial Cells—RNA in situ hybridizations were performed on different organs and sagittal sections of mice 1 day after birth with a specific probe for Slc5a8. We observed a strong expression of Slc5a8 in the submandibular gland, kidney, and gut at P1 (Fig. 1, B–D). In the adult kidney, we detected Slc5a8 mRNA in the kidney cortex, with more intense signals in the inner cortex (Fig. 1E). The related Na+-monocarboxylate transporter Slc5a12 was mainly detected in the outer cortex of the kidney (Fig. 1F) as previously described (13Srinivas S.R. Gopal E. Zhuang L. Itagaki S. Martin P.M. Fei Y.J. Ganapathy V. Prasad P.D. Biochem. J. 2005; 392: 655-664Crossref PubMed Scopus (105) Google Scholar). SLC5A8 expression was also investigated by Western blot analysis of multiple organs with an affinity-purified antiserum against a peptide close to the C terminus of the protein. Specific signals were detected in kidney, salivary gland, and large intestine (Fig. 1I). We did not detect the protein in brain and heart (Fig. 1I), liver, lung, spleen, muscle, testis, and uterus (data not shown). Immunofluorescence staining using the affinity-purified antiserum revealed a strong expression in the kidney, intestine, thyroid, and salivary gland, but not in the brain. The domains of protein expression corresponded well with expression of the mRNA detected by in situ hybridization. The expression of SLC5A8 in the kidney was confined to the cortex and the outer stripe of the outer medulla (Fig. 2A). The presence of SLC5A8 was restricted to the brush border of proximal tubules as indicated by costaining with phalloidin-TRITC, which identifies the brush border of the proximal tubules. Almost all proximal tubules of the outer stripe of the outer medulla were labeled, whereas many proximal tubules of the outer kidney cortex are devoid of SLC5A8 protein. Similar observations were also made by Gopal et al. (10Gopal E. Fei Y.J. Sugawara M. Miyauchi S. Zhuang L. Martin P. Smith S.B. Prasad P.D. Ganapathy V. J. Biol. Chem. 2004; 279: 44522-44532Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) who described the expression of SLC5A8 in the S3 segment of the proximal tubule. The expression of SLC5A8 in the thyroid gland was limited to the apical membrane of follicular cells (Fig. 2B). In the parotid gland, we detected expression of SLC5A8 at the apical membranes of intercalated duct cells (Fig. 2C) and of the acini (not shown). In the submandibular gland, immunoreactivity was only found in the apical membrane of the serous acini while the mucosal acini of this mixed salivary gland were negative for SLC5A8 (Fig. 2D). In the digestive tract, a double staining of monocarboxylic acid transporter 1 (MCT1, SLC16A1) and SLC5A8 was carried out. SLC5A8 was present in the apical membrane of both the surface epithelium and in crypts, whereas MCT1 labeling was exclusively observed in the basolateral membrane of surface epithelium and crypts. The intensity of SLC5A8 staining increased from the small intestine to the colon (Fig. 2, E–I). The jejunum presented a very faint but specific apical signal (not shown), whereas no immunoreactivity for SLC5A8 was detected in the duodenum and stomach (not shown). The anti-MCT1 (SLC16A1) antibody intensely labeled the large intestine (Fig. 2, F–J) but not the small intestine (Fig. 2E). The specificity of signals was always confirmed by parallel processing of the respective Slc5a8-/- tissues (Fig. 3K).FIGURE 3Histological sections of the thyroid of Slc5a8-/-/Slc26a4-/- mice. Sections (12 μm) are stained with hematoxylin/eosin. A, WT; B, Slc5a8-/-; C, Slc26a4-/-; D, Slc5a8-/-/Slc26a4-/-. No differences in thyroid histology between the four genotypes were observed. P, parathyroid gland. The scale bar corresponds to 200 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) SLC5A8 and SLC26A4 Alone and in Combination Are Dispensable for Normal Function of the Thyroid Gland—To investigate a possible role for SLC5A8 in the transport of iodide in the thyroid gland we measured the function of the thyroid in Slc5a8-/- and in Slc5a8-/-/Slc26a4-/- compound mutant mice. Histological investigation of the thyroid of up to 2-year-old Slc5a8-/- animals did not reveal any changes in the size of the thyroid or any other obvious morphological abnormalities. The histological examination was repeated in 5-month-old animals from Slc5a8/Slc26a4 cross-breedings. As expected, we did not observe any changes in morphology of Slc5a8-/- or Slc26a4-/- thyroids (Fig. 3, A–C); neither did we observe any apparent pathological changes in the thyroidea of Slc5a8/Slc26a4 compound mutant mice (Fig. 3D). We also analyzed the concentration of T4 in all different genotypes; no significant differences of T4 concentrations were detected (WT/WT: 2.27 ± 0.33 μg/dl (n = 6); Slc26a4+/+/Slc5a8-/-: 2.56 ± 0.37 (n = 8); Slc26a4-/-/Slc5a8+/+: 2.06 ± 0.27 (n = 8); KO/KO: 2.8 ± 0.33 (n = 8)). The concentration of TSH in blood was determined for WT (160 ± 7.0) and Slc5a8-/- (166 ± 7.2 ng/ml serum; n = 25/23) and was not significantly different. For WT versus Slc5a8-/- we also measured the 125I- incorporation into the serum protein as a measure for thyroid iodide organification as the percentage bound 125I/unbound 125I- in serum after 1 h of incorporation. We did not detect any
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