Characterization of a Heart-specific Fatty Acid Transport Protein

Abstract

Fatty acids are a major source of energy for cardiac myocytes. Changes in fatty acid metabolism have been implicated as causal in diabetes and cardiac disease. The mechanism by which long chain fatty acids (LCFAs) enter cardiac myocytes is not well understood but appears to occur predominantly by protein-mediated transport. Here we report the cloning, expression pattern, and subcellular localization of a novel member of the fatty acid transport protein (FATP) family termed FATP6. FATP6 is principally expressed in the heart where it is the predominant FATP family member. Similar to other FATPs, transient and stable transfection of FATP6 into 293 cells enhanced uptake of LCFAs. FATP6 mRNA was localized to cardiac myocytes by in situ hybridization. Immunofluorescence microscopy of FATP6 in monkey and murine hearts revealed that the protein is exclusively located on the sarcolemma. FATP6 was restricted in its distribution to areas of the plasma membrane juxtaposed with small blood vessels. In these membrane domains FATP6 also colocalizes with another molecule involved in LCFA uptake, CD36. These findings suggest that FATP6 is involved in heart LCFA uptake, in which it may play a role in the pathogenesis of lipid-related cardiac disorders. Fatty acids are a major source of energy for cardiac myocytes. Changes in fatty acid metabolism have been implicated as causal in diabetes and cardiac disease. The mechanism by which long chain fatty acids (LCFAs) enter cardiac myocytes is not well understood but appears to occur predominantly by protein-mediated transport. Here we report the cloning, expression pattern, and subcellular localization of a novel member of the fatty acid transport protein (FATP) family termed FATP6. FATP6 is principally expressed in the heart where it is the predominant FATP family member. Similar to other FATPs, transient and stable transfection of FATP6 into 293 cells enhanced uptake of LCFAs. FATP6 mRNA was localized to cardiac myocytes by in situ hybridization. Immunofluorescence microscopy of FATP6 in monkey and murine hearts revealed that the protein is exclusively located on the sarcolemma. FATP6 was restricted in its distribution to areas of the plasma membrane juxtaposed with small blood vessels. In these membrane domains FATP6 also colocalizes with another molecule involved in LCFA uptake, CD36. These findings suggest that FATP6 is involved in heart LCFA uptake, in which it may play a role in the pathogenesis of lipid-related cardiac disorders. long chain fatty acid fatty acid transport protein Homo sapiens FATP Mus musculus FATP glutathione S-transferase white adipose tissue Under normal conditions 60–70% of the oxidative metabolism of the heart comes from the β-oxidation of fatty acids (1Bing R.J. Circulation. 1955; 12: 635-647Crossref PubMed Scopus (13) Google Scholar). Extraction of fatty acids complexed to albumin by the heart is very efficient, with as much as 70% of the LCFAs1 from the blood entering the myocytes during a single transition through the cardiac capillary system (2Ballard F.B. Danforth W.H. Naegle S. Bing R.J. J. Clin. Invest. 1960; 39: 717-730Crossref PubMed Scopus (90) Google Scholar). Albumin-bound fatty acids are generated in the heart from triglycerides contained in lipoprotein particles by the action of lipases. Lipoprotein lipase is bound to the luminal face of endothelial cells after secretion from the parenchymal cells of the heart (3Henson L.C. Schotz M.C. Harary I. Biochim. Biophys. Acta. 1977; 487: 212-221Crossref PubMed Scopus (20) Google Scholar). Unlike hepatocytes, which have direct contact with the blood, fatty acids in the heart must first traverse the endothelial cell barrier. It is unclear how the LCFAs in the blood are transported across the endothelial cells, but it is unlikely that the entire LCFA-albumin complex leaves the circulation, because albumin does not diffuse through the clefts between cardiac vascular endothelial cells in significant amounts (4Bassingthwaighte J.B. Noodleman L. van der Vusse G. Glatz J.F. Mol. Cell Biochem. 1989; 88: 51-58Crossref PubMed Scopus (51) Google Scholar). Once in the interstitial fluid between endothelial cells and myocytes, fatty acids are again bound to albumin and rapidly taken up by the muscle cells. Although it was originally believed that uptake of LCFAs into cardiac myocytes is purely mediated by diffusion across the sarcolemma (4Bassingthwaighte J.B. Noodleman L. van der Vusse G. Glatz J.F. Mol. Cell Biochem. 1989; 88: 51-58Crossref PubMed Scopus (51) Google Scholar, 5DeGrella R.F. Light R.J. Basic Res. Cardiol. 1985; 80 Suppl. 2: 107-110PubMed Google Scholar), later work has shown that transport is predominantly protein-mediated (6Binas B. Danneberg H. McWhir J. Mullins L. Clark A.J. FASEB J. 1999; 13: 805-812Crossref PubMed Scopus (241) Google Scholar, 7Sorrentino D. Stump D. Potter B.J. Robinson R.B. White R. Kiang C.L. Berk P.D. J. Clin. Invest. 1988; 82: 928-935Crossref PubMed Scopus (157) Google Scholar, 8Stremmel W. J. Clin. Invest. 1988; 81: 844-852Crossref PubMed Scopus (180) Google Scholar, 9Schaffer J.E. Lodish H.F. Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (744) Google Scholar). A fatty acid transport protein (FATP) was identified by expression cloning from a murine adipocyte cDNA library as a protein that facilitates the uptake of LCFAs when overexpressed in adipocytes (9Schaffer J.E. Lodish H.F. Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (744) Google Scholar). This protein, later renamed FATP1 (10Hirsch D. Stahl A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8625-8629Crossref PubMed Scopus (374) Google Scholar), is induced during adipocyte differentiationin vitro and is expressed in fat tissue and in the brain, heart, kidneys, and skeletal muscle but not the liver. Subsequently, we reported the discovery of a large family of FATPs characterized by the presence of an FATP signature sequence of 311 amino acids that is highly conserved among FATP family members (10Hirsch D. Stahl A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8625-8629Crossref PubMed Scopus (374) Google Scholar, 11Faergeman N.J. Di Russo C.C. Elberger A. Knudsen J. Black P.N. J. Biol. Chem. 1997; 272: 8531-8538Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). FATP expression patterns include proteins exclusively or predominantly expressed in the liver, kidneys, small intestine, or in white adipose tissue (WAT). All FATPs are integral membrane proteins (12Lewis S.E. Listenberger L.L. Ory D.S. Schaffer J.E. J. Biol. Chem. 2001; 276: 37042-37050Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 13Stahl A. Gimeno R.E. Tartaglia L.A. Lodish H.F. Trends Endocrinol. Metab. 2001; 12: 266-273Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), and detailed subcellular localization studies have been reported for FATP1 and FATP4. The intestinal FATP, FATP4, is localized to the apical side of enterocytes and mediates the uptake of dietary fatty acids (14Stahl A. Hirsch D.J. Gimeno R. Punreddy S. Ge P. Watson N. Kotler M. Tartaglia L.A. Lodish H.F. Mol. Cell. 1999; 4: 299-308Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). FATP1 is present in the heart but is also strongly expressed in adipose tissue where its subcellular localization can change on insulin stimulation from an intracellular perinuclear compartment to the plasma membrane (15Stahl A. Evans J.G. Pattel S. Hirsch D. Lodish H.F. Dev. Cell. 2002; 2: 477-488Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The mechanisms and requirements for LCFA uptake through FATPs are poorly understood. Acyl-CoA synthetase activity has been demonstrated for several FATPs (16Choi J.Y. Martin C.E. J. Biol. Chem. 1999; 274: 4671-4683Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 17Coe N. Smith A. Frohnert B. Watkins P. Bernlohr D. J. Biol. Chem. 1999; 274: 36300-36304Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), and it has been suggested that this activity is required for transport. This notion has been challenged, however, by the recent observation that catalytic and transport activities of the yeast FATP homologue, FAT1, can occur independently (18Zou Z. DiRusso C.C. Ctrnacta V. Black P.N. J. Biol. Chem. 2002; 277: 31062-31071Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In addition to FATPs, two other proteins have been implicated in myocardial LCFA uptake: the cytoplasmic heart-specific fatty acid-binding protein and CD36. The strongest support for the role of these proteins comes from data demonstrating that in both heart-specific fatty acid-binding protein (6Binas B. Danneberg H. McWhir J. Mullins L. Clark A.J. FASEB J. 1999; 13: 805-812Crossref PubMed Scopus (241) Google Scholar) and CD36 (19Coburn C.T. Knapp Jr., F.F. Febbraio M. Beets A.L. Silverstein R.L. Abumrad N.A. J. Biol. Chem. 2000; 275: 32523-32529Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar) null mice cardiac fatty acid uptake and use is substantially reduced. Here we show that a novel member of the FATP family, FATP6, is a functional fatty acid transporter with a heart-specific expression pattern. Further, FATP6 localizes to the sarcolemma of cardiac myocytes juxtaposed with blood vessels where it colocalizes with CD36. This fact suggests that a novel mechanism may exist for the uptake of LCFAs into cardiac myocytes using FATP6, thus potentially implicating the molecule in a broad range of physiological and pathological cardiac functions. Clones encoding human FATP6 were identified by a search of public data bases for sequences similar to murine FATP1–5 coding regions using the BLASTX algorithm (20Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar). A DNA fragment containing the entire hsFATP6 coding sequence was inserted into the mammalian expression vectors pMet7 or pIRES-neo (Clontech) for transient and stable transfections, respectively. Transient transfection assays were performed as described previously (10Hirsch D. Stahl A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8625-8629Crossref PubMed Scopus (374) Google Scholar). For stable transfections, cells that had taken up the DNA were selected with 1 mg/ml G418 (Invitrogen). BODIPY-fatty acid uptake assays using a fluorescence-activated cell sorter and 14C-labeled fatty acid uptake assays were performed as described previously (10Hirsch D. Stahl A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8625-8629Crossref PubMed Scopus (374) Google Scholar, 14Stahl A. Hirsch D.J. Gimeno R. Punreddy S. Ge P. Watson N. Kotler M. Tartaglia L.A. Lodish H.F. Mol. Cell. 1999; 4: 299-308Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Human heart samples were obtained from the University of Pittsburgh Medical Center. In situhybridizations were performed essentially as described previously (14Stahl A. Hirsch D.J. Gimeno R. Punreddy S. Ge P. Watson N. Kotler M. Tartaglia L.A. Lodish H.F. Mol. Cell. 1999; 4: 299-308Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Briefly, paraformaldehyde-fixed dehydrated sections were hybridized with 35S-radiolabeled (5 × 107 cpm/ml) cRNA probes generated from the open reading frame of FATP6. After hybridization, slides were washed and dehydrated. To detect the localization of mRNA transcripts, the slides were dipped in Kodak NBT-2 photoemulsion and exposed for 7 days at 4 °C followed by development with Kodak Dektol developer. Slides were counterstained with hematoxylin and eosin and photographed. Controls for the in situ hybridization experiments included the use of a sense probe, which showed no signal above background in all cases. Human mRNA blots were obtained from Clontech. Blots were probed with32P-labeled DNA probes generated by PCR from the 5′-untranslated region of FATP6 using the Rapid-Hyb buffer (Amersham Biosciences). Unfixed rhesus monkey or mouse heart was washed with Hanks' buffered salt solution containing 1 mm EDTA, infused with 2.3 m sucrose solution, and embedded in O.C.T. 4583 compound. The material was cut into thick sections (15–40 μm). The sections were washed in phosphate-buffered saline containing 1% bovine serum albumin, 10% fetal calf serum, and 1% normal donkey serum to block nonspecific binding. Primary and secondary antibodies were diluted in blocking solution and incubated for 1 h. The sections were mounted in 90% glycerol/phosphate-buffered saline containing 1 mg/ml paraphenylinediamine and examined with a Zeiss LSM10 confocal system. Anti-FATP6 serum was raised by immunization of rabbits with the last 90 C-terminal amino acids fused to GST. Serum was affinity-purified over protein A columns and found to have minimal cross-reactivity against other FATP family members. Monoclonal anti-caveolin 3 antibodies and anti-CD31 were purchased from BD Biosciences. The anti-CD36 monoclonal antibody was a generous gift from Dr. Maria Febbraio. We initially reported the 3′ sequence of a human FATP using of FATP1 against the public data bases (10Hirsch D. Stahl A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8625-8629Crossref PubMed Scopus (374) Google Scholar). the 3′ FATP6 we public data bases of human expressed sequence and identified a cDNA for FATP6. The cDNA was by and was found later to to a cDNA sequence in as The FATP6 cDNA for a acid protein with a of of the FATP6 protein with the sequences of all other human FATPs were using the A that FATP6 is a member of the FATP family and is to FATP6 is of a and that is from the FATP1 and we localized hsFATP6 to human hsFATP6 sequence and localization was also by the human which showed that the coding region of the is over on of hsFATP6 against the mouse we were to a murine The protein is to hsFATP6 and is also in on mouse from the 5′-untranslated region of the was to detect its expression using of human tissue FATP6 was strongly expressed in heart but was from the brain, liver, and 1 of FATP6 in the heart by of were found in the and not FATP6 mRNA was present at very in and 1 A tissue of murine revealed that is predominantly expressed in the heart in this 1 expression was in the 1 To the cell that FATP6, in situ were performed with human heart A sense showed a very background signal the showed a signal with over cells identified by as cardiac myocytes To the subcellular localization of FATP6, we raised an against the C-terminal of FATP6. of rhesus monkey hearts were with serum and anti-caveolin 3 antibodies 3 is highly expressed by cardiac myocytes and was as a plasma membrane Z. D.S. I. Lodish H.F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). microscopy showed FATP6 in all cardiac myocytes was because of the FATP6 and 3 with the FATP6 protein FATP6 but not 3 3 and FATP6 protein was found exclusively on the sarcolemma. Unlike the 3 however, FATP6 was not the membrane but was in of LCFA from the interstitial to the To FATP6 was on sarcolemma sections in the direct of blood we heart sections with FATP6 and a for endothelial cells, FATP6 expression was in the areas to the expression was in the areas of blood vessels A and This expression was at as shown in where a small of a blood is by FATP6 expression on the myocytes above and the blood This expression is with a role of FATP6 in the uptake of LCFAs from the interstitial into cardiac is localized to blood vessels. rhesus monkey heart sections were with anti-CD31 antibodies or an and an is shown in the The in A and and in To that FATP6, the other FATP family facilitates the uptake of long chain fatty we cells with mammalian expression vectors containing FATP4, or FATP6 into 293 cells cells were identified by and uptake of LCFA was as described previously (10Hirsch D. Stahl A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8625-8629Crossref PubMed Scopus (374) Google Scholar, 14Stahl A. Hirsch D.J. Gimeno R. Punreddy S. Ge P. Watson N. Kotler M. Tartaglia L.A. Lodish H.F. Mol. Cell. 1999; 4: 299-308Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). cell FATP6 were generated by the transfection and of 293 cells. FATP6 were the cell with the uptake the to up fatty acids after a because of fatty acid of 14C-labeled by of the stable FATP6 cell with uptake is shown in with uptake by cells FATP1 or the expression and that of FATP6 can uptake of LCFAs. We the of hsFATP6 in with and using cell FATP6 and FATP1 had a for and with FATP4, the major intestinal LCFA transporter of the FATPs the uptake of fatty acids with chain both FATP1 and FATP6 are expressed in the heart, we to To this we performed blots with amounts of and heart from mice to of amounts of FATP1 or FATP6 The blots were probed with FATP1 and FATP6 and were by FATP6 was from but was expressed in the FATP1 was in in the heart After the to the we were to the FATP1 and FATP6 in the heart FATP1 was expressed at heart mg/ml FATP6 was which that FATP6 is the predominant FATP in the both FATP6 and CD36 are plasma membrane proteins that have been implicated in the uptake of LCFAs into we to a between these To this we sections of mouse hearts with antibodies against FATP6 CD36 3 and a in sections of murine FATP6 was present in of the plasma membrane and of the sections with an monoclonal serum showed a similar and with on the sarcolemma and In 3 was the plasma of all myocytes and The of FATP6 and CD36 and showed that FATP6 was the sarcolemma. of FATP6 with CD36 was in the white The of FATP6, and and showed that all are present in these membrane white as by the of the white These data that members of the FATP family may with CD36. Fatty acids are a major source of energy for cardiac myocytes, and in fatty acid metabolism have been implicated in cardiac disease. The mechanism by which fatty acids from the interstitial enter myocytes is not well but it has been that the of this uptake protein-mediated transport (6Binas B. Danneberg H. McWhir J. Mullins L. Clark A.J. FASEB J. 1999; 13: 805-812Crossref PubMed Scopus (241) Google Scholar, 7Sorrentino D. Stump D. Potter B.J. Robinson R.B. White R. Kiang C.L. Berk P.D. J. Clin. Invest. 1988; 82: 928-935Crossref PubMed Scopus (157) Google Scholar, 8Stremmel W. J. Clin. Invest. 1988; 81: 844-852Crossref PubMed Scopus (180) Google Scholar, 9Schaffer J.E. Lodish H.F. Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (744) Google Scholar). Here we report the cloning and of a novel member of the fatty acid transport protein family, termed FATP6, in and muscle other FATP family FATP1 as well as FATP6. FATP6 is however, FATP1 is expressed in a of other skeletal and the (9Schaffer J.E. Lodish H.F. Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (744) Google Scholar, D. Stahl A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8625-8629Crossref PubMed Scopus (374) Google Scholar, Martin G. P. M. J. H. J. 2000; PubMed Google Scholar). the notion that FATP6 is the predominant cardiac FATP is that the protein is FATP1 in mouse heart The heart also has a very uptake of fatty acids and in with a for with W. A. Res. 1985; PubMed Scopus Google Scholar). this is by the LCFA uptake of the cell which is with the that a significant of the cardiac LCFA uptake is mediated by FATP6. acid sequence of the FATP family show that human FATP6 is to a fatty acid transporter predominantly expressed in the and is the of a previously identified by as a very long chain A. N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This notion was by the that can as a fatty acid transporter (10Hirsch D. Stahl A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8625-8629Crossref PubMed Scopus (374) Google Scholar). It is unlikely that FATP6 is a very long chain because studies of FATP6 distribution in cardiac myocytes demonstrated that FATP6 is exclusively localized to the plasma membrane of cells. the mechanism by which FATPs transport LCFAs across is poorly and long chain as well as very long chain activities have been demonstrated for several mammalian FATP and yeast FATP (16Choi J.Y. Martin C.E. J. Biol. Chem. 1999; 274: 4671-4683Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 17Coe N. Smith A. Frohnert B. Watkins P. Bernlohr D. J. Biol. Chem. 1999; 274: 36300-36304Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). the that uptake is to of by FATPs or by a A. Gimeno R.E. Tartaglia L.A. Lodish H.F. Trends Endocrinol. Metab. 2001; 12: 266-273Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). In this it is that recent studies of the yeast FATP (18Zou Z. DiRusso C.C. Ctrnacta V. Black P.N. J. Biol. Chem. 2002; 277: 31062-31071Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) have demonstrated that in can the fatty acid from the very long chain synthetase (18Zou Z. DiRusso C.C. Ctrnacta V. Black P.N. J. Biol. Chem. 2002; 277: 31062-31071Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) that two and long chain and very long chain activity and that these are conserved in and FATPs 1 through of murine and human FATP6 sequences with the other family members that both are also conserved in FATP6 not which suggests that these mammalian proteins also as LCFA in the of catalytic direct of long chain and very long chain activity of hsFATP6 by Watkins J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) showed no activity above background when the protein was overexpressed in cells. with the data this that activity is not required for LCFA FATP6 is exclusively to areas of the sarcolemma that are juxtaposed to This of FATP6 with the transporter which on to the sarcolemma is on the plasma membrane S. Proc. Natl. Acad. Sci. U. S. A. 88: PubMed Scopus Google Scholar), and the diffusion of with LCFAs. Although FATP to an in LCFA uptake, it is that in several proteins to uptake of fatty acids in the In addition to FATPs, several other proteins have been implicated in LCFA uptake by cardiac myocytes, CD36 and fatty acid-binding and it has been suggested that these proteins may to LCFA uptake A. Gimeno R.E. Tartaglia L.A. Lodish H.F. Trends Endocrinol. Metab. 2001; 12: 266-273Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Here we for the first that FATPs are in to CD36 on the plasma This fact the that may to LCFAs on the plasma membrane and on to FATPs for Changes in heart fatty acid metabolism have been to cardiac cardiac J. Sci. 1999; PubMed Google Scholar) and J. Biochim. Biophys. Acta. 1994; PubMed Scopus Google Scholar). Although normal hearts use fatty acids as energy hearts of with cardiac on metabolism by a in fatty acid J. Sci. 1999; PubMed Google Scholar, der Vusse van M. Glatz J.F. Res. 2000; PubMed Scopus Google Scholar). of the involved in fatty acid use in the heart has in fact been shown to (6Binas B. Danneberg H. McWhir J. Mullins L. Clark A.J. FASEB J. 1999; 13: 805-812Crossref PubMed Scopus (241) Google Scholar, A. H.F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Fatty acid use can with from and that block fatty acid as and the of after in J. Biochim. Biophys. Acta. 1994; PubMed Scopus Google Scholar). A recent FATP1 expression in hearts with hearts from with found no in mRNA for this transporter J. A. R. W. J. Res. 2000; Google Scholar). FATP6 expression was not It of to FATP6 is during the development of cardiac and or of the transporter can cardiac disease. We Dr. D. for reading and on this the University of Pittsburgh Medical for human heart for with for with fluorescence-activated cell sorter and at the for the localization of the FATP