Lipid transporters: membrane transport systems for cholesterol and fatty acids.

Lipophilic molecules can passively diffuse across cell membranes, a process that is driven by the concentration gradient, by availability of acceptors to facilitate desorption from the bilayer, and by cellular metabolism. However, evidence has accumulated that supports the existence of specialized, protein-facilitated membrane transport systems for many lipophilic molecules. This has generated considerable debate regarding why such systems need to exist. The present review summarizes recent developments related to the membrane transport systems for cholesterol and fatty acids, which have been shown to involve structurally related proteins. General similarities of the cholesterol and fatty acid systems to other lipid transport systems (briefly discussed in the Introduction section) are highlighted in the Conclusion section. The overall aim of the present review is to illustrate why lipid transporters are needed in vivo, and how they accomplish specific functions that can not be met by lipid diffusion alone.

CD36 antisense expression in 3T3-F442A preadipocytes.

An adipocyte membrane glycoprotein, FAT, homologous to CD36, has been implicated in the binding/transport of long-chain fatty acids. FAT/CD36 was identified by reaction with reactive long chain fatty acids derivatives under conditions where they inhibited FA uptake. Expression of CD36 in fibroblasts lacking the protein led to induction of a saturable high affinity, phloretin-sensitive component of oleate uptake. In this report, we have examined the effects of FAT/CD36 antisense expression in 3T3-F442A preadipocyte cells, on FA uptake and cell differentiation. Cells were transfected with pSG5-TAF vector obtained by insertion of antisense coding sequence of FAT/CD36 into the BamH 1 site of pSG5. Four clones were selected based on expression of antisense CD36 mRNA. Levels of CD36 protein were determined by flow cytometry and correlated with rates of oleate uptake. Three clones, TAF13, TAF25, and TAF38 exhibited low CD36 expression and one clone TAF 18 had expression comparable to that of F442A control cells. FA uptake rates in clones TAF13, TAF25 and TAF38 were lower than those observed in TAF18. At confluence, adipocyte differentiation could be promoted by addition of insulin and triiodothyronine only in TAF18 cells but not in TAF13, TAF25 or TAF38. Addition of fatty acids to clones TAF13, TAF25 and TAF38 lead to an induction of CD36 expression, an enhancement of FA uptake and better cell differentiation. The data support a role of CD36 in the membrane uptake of long chain FA. CD36 expression and FA uptake appear to be closely linked to preadipocyte differentiation.

Regulation of FAT/CD36 gene expression: further evidence in support of a role of the protein in fatty acid binding/transport.

Much biochemical evidence has implicated rat adipocyte CD36 (FAT) in membrane binding and transport of long-chain fatty acids (FA). Expression of the mRNA favored tissues with active FA metabolism and was upregulated in vivo with diabetes and with high fat feeding. In culture, CD36 mRNA was a strong marker of preadipocyte differentiation and was modulated by the same factors effective on mRNAs coding for other proteins involved in FA metabolism. In preadipocytes, long-chain FA or 2-bromopalmitate but not short-chain FA strongly induced CD36 mRNA within 8 h to an optimum within 24 h. Removal of the FA resulted in a decay of CD36 mRNA with a half life of about 12 h. In differentiated adipocytes, levels of CD36 mRNA were downregulated by the 3': 5'-cyclic adenosine monophosphate, cAMP, analog, 8-(4-chlorophenylthio) adenosine, 8-CPT, at concentrations of 1-100 microM. The effect, observed within 6 h, was optimal after 18 h and independent of the action of 8-CPT to mobilize FA. Regulation of CD36 expression by factors effective on expression of other proteins implicated in FA metabolism is consistent with its role in membrane FA transport.

Expression of the CD36 homolog (FAT) in fibroblast cells: effects on fatty acid transport.

An adipocyte membrane glycoprotein, (FAT), homologous to human CD36, has been previously implicated in the binding/transport of long-chain fatty acids. It bound reactive derivatives of long-chain fatty acids and binding was specific and associated with significant inhibition of fatty acid uptake. Tissue distribution of the protein and regulation of its expression were also consistent with its postulated role. In this report, we have examined the effects of FAT expression on rates and properties of fatty acid uptake by Ob17PY fibroblasts lacking the protein. Three clones (P21, P22, and P25) were selected based on FAT mRNA and protein levels. Cell surface labeling could be demonstrated with the anti-CD36 antibody FITC-OKM5. In line with this, the major fraction of immunoreactive FAT was associated with the plasma membrane fraction. Assays of oleate and/or palmitate uptake demonstrated higher rates in the three FAT-expressing clones, compared to cells transfected with the empty vector. Clone P21, which had the highest protein levels on Western blots, exhibited the largest increase in transport rates. Fatty acid uptake in FAT-expressing P21 cells reflected two components, a phloretin-sensitive high-affinity saturable component with a Km of 0.004 microM and a basal phloretin-insensitive component that was a linear function of unbound fatty acid. P21 cells incorporated more exogenous fatty acid into phospholipids, indicating that binding of fatty acids was followed by their transfer into the cell and that both processes were increased by FAT expression. The data support the interpretation that FAT/CD36 functions as a high-affinity membrane receptor/transporter for long-chain fatty acids.