The Journal of Nutrition Commentary

Lipid RaftsComposition, Characterization, and Controversies

Philip C. Calder1* and Parveen Yaqoob2
1 Institute of Human Nutrition, School of Medicine, University of Southampton, Southampton S016 7PX, UK and 2Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, University of Reading, Reading RG6 6AP, UK

See related article: J Nutr. 137-3: 548553, 2007.

In this issue of The Journal of Nutrition, an article reporting on the effect of (n-3) PUFA on the composition of lipid rafts in tumor cells is published, to our knowledge, for the rst time (1). The authors report that the enrichment of lipid rafts of MDAMB-231 breast cancer cells with (n-3) PUFA is probably linked to modication of cell signaling processes leading to cell death. The results of this study further our understanding of the mechanism by which different types of fatty acid can inuence tumor cell growth. So, what are lipid rafts, and why does this article represent an important step forward in our knowledge? Lipid rafts are regions of membranes with a distinct, characteristic structural composition and that appear to act as platforms to colocalize proteins involved in intracellular signaling pathways. The organization of membranes into such microdomains recognizes that, far from being randomly arranged, lipids may actually be highly organized within different parts of the membrane, and that this organization inuences the way that membrane proteins are distributed (2). Rafts are particularly rich in sphingolipids and cholesterol, and the side chains of the phospholipids present are usually highly enriched in saturated fatty acids compared with the surrounding nonraft regions of the membrane. Enrichment of phospholipids with saturated fatty acids allows for the close packing of lipids within rafts because 1) sphingolipids also contain saturated fatty acid side chains, and 2) cholesterol and saturated fatty acids are able to pack closely. As a result of the presence of cholesterol and saturated fatty acids, lipid rafts are more ordered and less uid than the surrounding membrane. Cytoplasmic proteins that are covalently modied by saturated fatty acids (palmitoyl or myristoyl moieties) and cell surface proteins that are attached via a glycosyl phosphatidylinositol anchor are highly concentrated within lipid rafts. Many proteins involved in signal transduction, such as Src family kinases, G proteins, growth factor receptors, mitogen-activated protein kinase (MAPK),3 and protein kinase C are predominantly found in lipid rafts, which appear to act as signaling platforms by bringing together (i.e., colocalizing) various signaling components, facilitating their interaction (3). Despite a large body of work, doubts still persist regarding the existence and nature of lipid rafts (47). These doubts have
3 Abbreviations used: DHA, docosahexaenoic acid; EGF, epidermal growth factor; EPA, eicosapentaenoic acid; MAPK, mitogen-activated protein kinase. * To whom correspondence should be addressed. E-mail: pcc@soton.ac.uk.

arisen mainly because of limitations in the methods available to isolate and to study rafts. Lipid rafts have been dened largely according to their insolubility in nonionic detergents such as Triton X-100, which probably relates to their structural characteristics, and they have frequently been termed detergentresistant membranes on this basis. Detergent-resistant membranes have traditionally been isolated by density gradient otation of cellular material that has been homogenized in the presence of Triton X-100. Flotillin and glycosyl phosphatidylinositol anchored proteins are used as raft markers, and the material obtained in this way is rich in cholesterol and sphingolipids. However, variation in experimental conditions can have a major impact on exactly what is obtained in the detergent-resistant fraction. For example, detergents other than Triton X-100 can be used, and the nature of the material that is isolated depends upon the detergent used and its concentration. Thus, different methods yield different membrane fractions, and so the nature of the membrane material (rafts) studied by different investigators varies. Clearly, direct visualization of rafts, for example by microscopy, would resolve some of the uncertainties about their existence and structure, but uorescence microscopy studies have tended to produce mixed results (4). Membrane microdomains have been studied extensively with respect to T-lymphocyte responses to activation (810). Furthermore, T cells are the only cell type in which the effects of PUFA on the structure and function of detergent-resistant membranes have been studied to any great extent. PUFA have long been known to alter T cell functional responses (11), and research over the last few years suggests that raft disruption underlies the mechanism of action of PUFA upon T cells (1217). Schley et al. (1) report the effect of (n-3) PUFA on the composition of detergent-resistant membranes (which they term lipid rafts) in the MDA-MB-231 human breast cancer cell line. The study by Schley et al. (1) appears to be the rst examination of the inuence of fatty acids on detergent-resistant membranes in tumor cells and furthers our understanding of the mechanism by which different types of fatty acid can inuence tumor cell growth. The cells were grown in a medium supplemented with linoleic acid or the long-chain (n-3) PUFA, eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA), or combinations of linoleic acid and long-chain (n-3) PUFA. The (n-3) PUFA were shown to decrease cell numbers, as previously observed by others with this cell line. Detergent-resistant membranes were
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0022-3166/07 $8.00 2007 American Society for Nutrition. J. Nutr. 137: 545547, 2007. Manuscript received 20 December 2006.

isolated by a standard procedure involving density gradient separation of cellular material homogenized in the presence of cold Triton X-100. In accordance with the expected characteristic of lipid rafts, the detergent-resistant membranes had a higher content of saturated fatty acids (; 100% higher) and a lower content of PUFA (; 50% lower) than total membrane phospholipids. Detergent-resistant membranes also contained less EPA and DHA than bulk membrane phospholipids. Incubation with EPA and DHA resulted in increased concentrations of these 2 fatty acids in both bulk membrane phospholipids and in detergent-resistant membranes, with the enrichment impaired by the inclusion of linoleic acid in the medium. The extent of (n-3) PUFA enrichment was lower in detergent-resistant membranes than in bulk membranes. Incubation with EPA and DHA altered the composition of detergent-resistant membranes: there was less sphinogomyelin (25% less) and cholesterol (40% less) and more phosphatidycholine (15% more). The observation of decreased sphingomyelin is consistent with ndings for detergentresistant membranes from T cells of (n-3) PUFA-fed mice (16). Altered content of lipids within detergent-resistant membranes may reect perturbed movement of lipids between detergentresistant membranes and the bulk membrane or it may reect hydrolysis of lipid within detergent-resistant membranes. For example, decreased sphingomyelin content may relate to hydrolysis by sphinogomyelinase, which would generate ceramide. Indeed, Schley et al. (1) report a higher ceramide content in detergent-resistant membranes from cells treated with EPA and DHA. Wu et al. (18) reported that EPA and DHA increased neutral sphingomyelinase activity in MDA-MB-231 cells. It is intriguing to note that neutral sphingomyelinase is believed to be localized to lipid rafts. Ceramide has been shown to induce apoptosis, and thus an enhancement of neutral sphinomyelinase activity as a result of a change in lipid raft fatty acid composition may be the mechanism by which PUFA inuence tumor cell viability. Thus, long -chain (n-3) PUFA may modify both the nature of the lipid present in rafts and its fatty acid composition. Altered lipid (e.g., sphingomyelin, cholesterol) content of rafts would be expected to alter raft function. Incubation with EPA and DHA decreased epidermal growth factor (EGF) receptor levels in detergent-resistant membranes but not in whole cells (1). Despite lower levels of EGF receptor in detergent-resistant membranes, total cell phosphorylated EGF receptor was increased by (n-3) PUFA. This is consistent with the loss of EGF receptor from rafts followed by its phosphorylation, a process believed to occur upon EGF receptor migration out of rafts. Cholesterol depletion causes loss of EGF receptor from lipid rafts and promotes its binding to EGF and phosphorylation. The (n-3) PUFAs also increased the amount of cellular phosphorylated p38 MAPK, which is downstream from the EGF receptor, but total MAPK was not affected (1). Although the observation of increased EGF receptor phosphorylation and activity seems to oppose the idea that EGF promotes cell proliferation and survival, there is evidence that the EGF receptor is associated with apoptosis in MDA-MB-231 cells (19). Indeed, p38 MAPK activation, as seen by Schley et al. (1), promotes tumor cell apoptosis (20). Thus, the overall picture that emerges from Schley et al. (1) is that long-chain (n-3) PUFA are incorporated into detergent-resistant membranes of cultured breast cancer cells where they act to alter the nature of the lipid present. This may be through activation of neutral sphingomyelinase, so depleting the membrane of sphingomyelin, and through cholesterol exclusion. Sphingomyelin hydrolysis generates ceramide, a proapoptotic signal, whereas the lowered content of cholesterol results in cytosolic expulsion of the EGF receptor. Subsequent
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phosphorylation of the latter generates further proapoptotic signals, perhaps through p38 MAPK activation. Ultimately, these events lead to cell apoptosis, as described elsewhere for this cell line, and reduced cell numbers, as reported here (1) and previously. Clearly, the data presented by Schley et al. (1) are important and make a novel contribution to our understanding of how (n-3) PUFA interact with detergent-resistant membranes and how they act to reduce breast cancer cell growth. This work highlights membrane microdomains as the focus for improved understanding of membrane-mediated signaling events and of the effects of fatty acids and other lipids on cell growth and function. However, it is important to note that Schley et al. (1) used a specic procedure to isolate detergent-resistant membranes (this is essentially the same Triton X-100 based procedure used by others working with (n-3) PUFA and detergent-resistant membranes in T cells (1217)), and, as noted earlier, different methodologies generate membrane material with differing structural characteristics. Improved techniques for the visualization of lipid rafts in intact, living cells would greatly aid advances in this area. Until such visualization is possible, doubts about the true nature of lipid rafts remain. However, if what has been demonstrated in studies of PUFA and detergent-resistant membrane structure and function (1217), including these new data of Schley et al. (1), is true, then we may, at last, be closer to understanding the mechanism by which these fatty acids inuence the activity of so many different cell types.

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