FAT/CD36 expression alone is insufficient to enhance cellular uptake of oleate Nicholas S. Eyre a , Leslie G. Cleland b , Graham Mayrhofer a,b, * a School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia b Arthritis Research Laboratory, Hanson Institute for Medical Research, Institute of Medical and Veterinary Science, Adelaide, SA, Australia article info Article history: Received 13 February 2008 Available online 24 March 2008 Keywords: FAT/CD36 Long-chain fatty acid transport Lipid rafts Caveolae Tet-off abstract Fatty acid translocase (FAT/CD36) is one of several proteins implicated in receptor-mediated uptake of long-chain fatty acids (LCFAs). We have tested whether levels of FAT/CD36 correlate with cellular oleic acid import, using a Tet-Off inducible transfected CHO cell line. Consistent with our previous findings, FAT/CD36 was enriched in lipid raft-derived detergent-resistant membranes (DRMs) that also contained caveolin-1, the marker protein of caveolae. Furthermore in transfected cells, plasma membrane FAT/CD36 co-localized extensively with the lipid raft-enriched ganglioside GM1, and partially with a caveolin-1- EGFP fusion protein. Nevertheless, even at high levels of expression, FAT/CD36 did not affect uptake of oleic acid. We propose that the ability of FAT/CD36 to mediate enhanced uptake of LCFAs is dependent on co-expression of other proteins or factors that are lacking in CHO cells. Crown Copyright Ó 2008 Published by Elsevier Inc. All rights reserved. Cellular acquisition of long-chain fatty acids (LCFAs) is thought to involve two mechanisms. The first is passive diffusion or ‘flip- flop’ of LCFAs across the plasma membrane [1]. The second in- volves protein-mediated import of LCFAs [2]. This latter pathway is probably of greatest significance when levels of free fatty acid (FFA) are low (e.g. during fasting) and/or when the metabolic requirements for LCFAs are high. Several plasma membrane trans- porters of LCFAs have been identified, namely FAT/CD36, plasma membrane fatty acid binding protein (FABPpm) and six isoforms of fatty acid transport protein (FATP) [3]. FAT/CD36 is the best characterized and FAT/CD36-knockout mice display changes in LCFA utilization and metabolism consistent with an important role for the molecule in LCFA transport [4,5]. Recent studies have demonstrated that localization of FAT/CD36 to plasma membrane lipid rafts/caveolae is critical for activity in LCFA transport [6,7]. Firstly, depletion of these cholesterol- and sphingolipid-rich membrane microdomains with the cholesterol- binding agent methyl-b-cyclodextrin, and specific inhibition of FAT/CD36 with sulfosuccinimidyl oleate (SSO), results in non-addi- tive inhibition of oleate uptake in 3T3-L1adipocytes [6]. Further- more, delivery of FAT/CD36 to the plasma membrane and LCFA uptake is restricted in embryonic fibroblasts prepared from caveo- lin-1-null mice [7]. Expression of recombinant caveolin-1-EGFP re- instated formation of morphological caveolae in these cells and caused both redistribution of FAT/CD36 to the plasma membrane and increased uptake of oleic acid. However, dependence of FAT/CD36 on caveolin-1 for localization to the plasma membrane or for activity in LCFA uptake is not universal [8]. We show that although FAT/CD36 is located in the plasma membrane and enriched in lipid rafts/caveolae in transfected CHO cells, expression over a range of levels did not enhance uptake of oleic acid. We postulate that CHO cells lack a partner protein(s) or other factors required for FAT/CD36 to enhance cellular impor- tation of LCFAs. Materials and methods Materials. Cell culture reagents were purchased from Gibco BRL. Fetal bovine serum (FBS) was purchased from Trace Biosciences. FuGene6 transfection reagent and molecular biology reagents were purchased from Roche Applied Science. Puro- mycin dihydrochloride, doxycycline, mammalian protease inhibitor cocktail, phloretin, fatty acid-free BSA and oleic acid were from Sigma. Plasmids pEF-IRES- puro 6 and pSG5-FAT were gifts from Dr. Daniel Peet (University of Adelaide, Ade- laide, Australia) and Dr. Nada Abumrad (Washington University, St. Louis, USA) respectively. Alexa 594-conjugated cholera toxin B subunit (CT-B) was from Molec- ular Probes. The anti-FAT/CD36 monoclonal antibodies UA009 and MO25 have been described previously [9,10], the latter a gift from Dr. Narendra Tandon (Otsuka Maryland Medicinal Laboratories, Rockville, USA). The following antibodies were purchased: mouse monoclonal anti-caveolin-1 (BD Transduction Laboratories), mouse monoclonal anti-b-actin (Sigma), horseradish peroxidase-conjugated anti- mouse IgG F(ab 0 ) 2 (Amersham Biosciences), FITC-conjugated anti-mouse immuno- globulin (BD Pharmingen), and Cy3-conjugated anti-mouse IgG (Jackson Immuno- Research Laboratories). SuperSignal West Femto chemiluminescence substrate was purchased from Pierce Biotechnologies. [ 14 C]-oleic acid was purchased from Amersham Biosciences. Cell culture. Chinese hamster ovary (CHO) cells were grown in RPMI medium supplemented with 10% FBS, 2 mM glutamine, 10 lg/ml streptomycin, and 10 units/ml penicillin in a 5% CO 2 /95% air atmosphere at 37 °C. Following initial selection, stable transfectants were maintained in medium containing G418 (500 lg/ml) and puromycin (3 lg/ml). Doxycycline was added to a final concentra- tion of 0.01–100 ng/ml, as indicated. 0006-291X/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.02.164 * Corresponding author. Address: School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia. Fax: +61 8 8303 7532. E-mail address: graham.mayrhofer@adelaide.edu.au (G. Mayrhofer). Biochemical and Biophysical Research Communications 370 (2008) 404–409 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc