FAT/CD36 expression alone is insufficient to enhance cellular uptake of oleate

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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.

Section snippets

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. Puromycin 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, Adelaide, Australia) and Dr.

Generation of CHO cell lines displaying doxycycline-sensitive expression of FAT/CD36

Cloned CHO cells expressing the tetracycline-controlled transactivator (tTA) protein (clone 1.2) were stably transfected with the tTA-responsive FAT/CD36 expression plasmid pTRE2-FAT/CD36-IRES-puro. Clones of puromycin-resistant double transfectants were isolated and screened by flow cytometry for expression of FAT/CD36 and for sensitivity of FAT/CD36 expression to doxycycline (100 ng/ml). Clone C3 was chosen, as it displayed high levels of un-repressed FAT/CD36 expression and sensitivity to

Discussion

Involvement of FAT/CD36 in protein-mediated transport of LCFAs has received strong support from studies in FAT/CD36-knockout mice and mice with muscle-specific expression of FAT/CD36 [20]. Furthermore, rapid translocation of FAT/CD36 from intracellular stores to the plasma membrane has been observed in response to acute stimuli (such as insulin treatment and contraction in muscle cells), and these changes correlate with increases in LCFA acid uptake [21]. Unexpectedly, we did not find a

Acknowledgments

The authors thank Dr. Meredith Wallwork (University of Adelaide, Adelaide, Australia) for generous assistance with confocal microscopy and Rebecca Fitzsimmons for helpful discussions.

References (28)

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    If this were the case, the result would be the simultaneous lipolytic stimulation from the catecholamine, and anti-lipolytic action from the resultant peroxide, a physiologically unlikely possibility. On the other hand, although the function of CD36 as a FA transporter is controversial [79], mice engineered to lack this protein are hyperlipidemic and show abnormalities in lipoprotein uptake [80]. In fact, the phenotype of the CD36 null mouse is not dissimilar from that of a cavin-1 or a caveolin-1 deficient mouse, and indeed, the aortas of this last animal have greatly diminished CD36 expression [81].

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