Effluxed lipids: Tangier Island’s latest export

In 1608, the intrepid English explorer, Captain John Smith, set out to map the new colony of Virginia and its Chesapeake Bay environs (1). Approximately 20 miles west of the Eastern Shore, he encountered three islands that he named the Russells after his ship’s surgeon, Dr. William Russell. Today, one of the islands bears Smith’s name and another is called Tangier Island because, according to local lore, its sandy shores reminded the captain of the white dunes of the port of Tangier, Morocco on the Strait of Gibraltar (2). For most of the past 400 years, the inhabitants of Tangier Island have been both economically sustained and genetically insulated by the bay that surrounds them. A calamitous outbreak of cholera in 1866 led to the evacuation and quarantine of the island, and its subsequent repopulation by a much smaller number of the displaced islanders added to the genetic insularity of its inhabitants. Most of the island’s current population are descendants of this small group of hardy individuals, with more than 600 of the approximately 800 residents bearing the surname Crockett, Pruitt, or Parks (2). It is not surprising, therefore, that when another explorer, Donald Fredrickson of the National Institutes of Health, traveled to Tangier in 1960 to find individuals who shared the phenotype of orange (cholesterol ester-laden) tonsils that he and his colleagues had found in a young boy from the island, the one similar individual identified was the boy’s younger sister (3). The findings of orange, lipid-engorged tonsils and nearly absent high density lipoprotein (HDL) cholesterol levels, shared by the siblings, are the hallmarks of the disorder to which the island has given its name, Tangier Disease (4).

In the past decade, studies of fibroblasts taken from patients with Tangier Disease have demonstrated a defect in the export of cholesterol from these cells to the major protein constituent of HDL, apolipoprotein AI (apoAI) (5–7). The failure of cells to efflux cholesterol to HDL in vivo would lead to a lipid-depleted HDL in the circulation. Metabolic turnover studies have shown that such lipid-poor HDL are very rapidly cleared from the blood, probably by the kidney (8, 9). The failure to move cellular cholesterol to HDL therefore would account, at least in part, for the low levels of HDL and HDL cholesterol seen in Tangier patients. Although these observations have delineated the general metabolic problem in patients with Tangier Disease, the cell biologic explanation for the efflux abnormality has proven more refractory to our understanding. In this issue of the Proceedings, Takahashi and Smith (not to be confused with the seafaring captain) (10) report a novel mechanism through which apoAI appears to remove cholesterol from cells. It is this specific process of apolipoprotein-stimulated cholesterol efflux that is most clearly defective in individuals with Tangier Disease.

Cells have evolved very elegant mechanisms for controlling their cholesterol content. Although our understanding of these mechanisms is chiefly the result of work involving the de novo cholesterol synthesis and receptor uptake pathways whose activation increases cellular cholesterol content, there are also pathways for ridding cells of excess cholesterol (11). As cholesterol accumulates in cells, it can be stored in its acylated form in cytoplasmic lipid droplets that arise from the action of the cholesterol-esterifying enzyme, acyl-CoA-cholesterol-acyltransferase (ACAT). Mobilization of this stored cholesterol can be stimulated by incubating cells with apoAI. The cholesterol ester is hydrolyzed to unesterified cholesterol and then traffics to the plasma membrane via a route that is not well mapped. The apolipoprotein acceptor then acquires the cholesterol in a process that entails more than simple desorption and diffusion from the plasma membrane. If the acceptor is apoAI in a nascent HDL particle, the cholesterol can be re-esterified by the action of an associated enzyme, lecithin cholesterol acyl transferase (LCAT), and then stored in the nonpolar lipid core of the HDL particle. This process triggers what often is referred to as the reverse cholesterol transport pathway and has long been postulated to represent the physiologic basis for the association of elevated HDL cholesterol levels with lower rates of coronary heart disease (12). By removing cholesterol from the cells in which it chiefly accumulates in atherosclerotic lesions (i.e., the monocyte/macrophages), the reverse cholesterol transport pathway provides a means by which the artery wall can protect itself from unwanted lipid deposition.

In previous work, Smith’s laboratory showed that a mouse macrophage cell line, RAW 264, had a cAMP-inducible increase in cholesterol efflux to another lipid acceptor apolipoprotein, apoE (13). Treatment with the cAMP analogue, 8-Br-cAMP, increased the binding of both apoE and apoAI to the treated RAW cells, suggesting that increased adenylate cyclase activity led to greater expression, or conformational activation, of the plasma membrane protein responsible for tethering the apolipoproteins to the cell. As the identity of this docking protein is unknown, it is a reasonable candidate for the protein that is defective in Tangier Disease. In the current paper, the authors examined the one established HDL receptor, SR-BI (14), for cAMP responsiveness and found a substantial decrease in its expression, making it unlikely to be the protein responsible for enhanced binding. More interestingly, the authors found that cAMP treatment resulted in a dramatic increase in the internalization of radiolabeled apoAI and a subsequent resecretion of 58% of the cell-associated label. Although, SR-BI mediates lipid transfer from HDL into cells, the available evidence indicates that this receptor’s activity does not result in the internalization of the protein component of the lipoprotein. Thus, it would appear that a novel protein is required for this action. The authors also present evidence that cholesterol is released from the cells at the same time the apolipoprotein re-emerges, suggesting that internalized apoAI carries the cholesterol out with it upon resecretion. Finally, using a variety of cell biologic methods, the authors provide evidence that indicates that a calcium-dependent endocytosis pathway is involved in the process. Several years ago, Schmitz et al. (15) described a pathway of HDL uptake and resecretion, termed retroendocytosis, that is quite similar to what Takahashi and Smith now report for the apolipoprotein component alone. Although this earlier observation concerning HDL was controversial (16), the technical difficulties of performing these experiments may account for the discrepant results. The current work strengthens the evidence that the uptake of HDL or its apolipoprotein component may be necessary for efficient cholesterol efflux. Nevertheless, there are some caveats that must be mentioned in the interpretation of these studies.

The work by Takahashi and Smith was done by using a transformed mouse monocyte cell line. The actual quantitative impact of resecretion on total cholesterol efflux from these cells is difficult to assess in the data presented. In most laboratories, including our own, cholesterol efflux from lipid-loaded human fibroblasts typically represents 10–20% of the labeled cellular sterol, an amount that may be substantially higher than that arising from the resecretion pathway in RAW cells. As human fibroblasts do not require cAMP treatment to export this amount of cholesterol to apolipoprotein acceptors, it is not clear if the RAW response to cAMP involves stimulation of a specific mechanism that is common to the fibroblast and macrophage efflux pathways, or if some more general effect on macrophage function accounts for the change. Finally, some of Takahashi and Smith’s data implicating endocytosis in the efflux pathway depend on chemical inhibitors whose effects may not be confined to the endocytotic events they postulate to be involved in apoAI uptake and resecretion. Despite these caveats, the data provides an intriguing insight into cholesterol efflux and the mechanism by which apoAI may stimulate it. Clearly, the identification of the protein responsible for mediating the enhanced apoAI binding that results from cAMP stimulation would seem to be the logical next step in the characterization of this pathway. Remarkably, that may have already occurred.

Over the summer, several laboratories independently identified and three groups have now published studies demonstrating that an ATP binding cassette (ABC) transporter is mutated in patients with Tangier Disease (17–19). This ABC1 transporter, originally cloned by Chimini and colleagues (20, 21) using PCR amplification based on homology to other ABC proteins, is a widely expressed, putative 12-membrane spanning protein, whose activity in macrophages is up-regulated by sterol loading. The protein adds to the growing list of ABC family members linked to human diseases, several of which involve errors in lipid handling (22–24). The papers linking this protein to Tangier Disease detail the localization of the gene within a previously mapped region of chromosome 9 (25). These reports also describe multiple mutations, several of which would clearly result in a nonfunctional protein. The studies do not, however, contain functional data that indicate that apoAI directly binds to the ABC1 transporter. So, it is by no means certain that the binding protein that Smith’s laboratory finds to be up-regulated by cAMP treatment of RAW cells will prove to be the murine ortholog of human ABC1. The protein does increase iodide transport in response to cAMP, however, and Becq et al. (26) have shown that protein kinase A phosphorylates it in vitro. The dependence of cholesterol efflux on PKA phosphorylation of this protein undoubtedly will be examined in short order. As there are other structural elements within the ABC1 protein that suggest interactions with different membrane-associated proteins, future studies also could show that these as yet unidentified proteins are responsible for the apoAI binding or cAMP regulation that Smith’s lab has identified in the RAW cells.

The discovery of the ABC1 transporter’s link to abnormal cholesterol efflux promises to lead to a host of new insights into lipid metabolism. Previous work implicating protein kinase C and phospholipases C and D activation in cholesterol efflux pathways now can be reconsidered in light of their effects on ABC1 (27, 28). The role of ABC1 in phospholipid efflux also can be explored. Recent work on a highly homologous ABC transporter, Rim, involved in phosphatidylethanolamine transport in rod photoreceptor outer segments, suggests that exploration will be a fruitful one (29). From a clinical standpoint, the inverse relationship between HDL levels and coronary artery disease makes the cholesterol efflux pathway and the ABC1 transporter potential targets for therapeutic agents designed to improve cholesterol removal from atherosclerotic plaques (30). Whether the many individuals with more modestly reduced HDL cholesterol levels and coronary heart disease also will have defects in the ABC1 transporter pathway is a question likely to engage the interest of epidemiologists and geneticists (31, 32). Finally, the enormous impact of ABC1 on the serum lipid profile, as evidenced by Tangier patients’ dramatically reduced HDL and low density lipoprotein cholesterol levels, indicates that an understanding of this gene’s function will profoundly affect our knowledge of serum lipoprotein metabolism.

It may be too much to assume that the spirit of Captain Smith, renowned for his irascibility, truculence, and conceit, is smiling at the work of the latest explorers to put Tangier Island again on the world’s map. However, one suspects that a grudging respect for the genetic mapmakers would be forthcoming, as they, too, have helped open a new world whose further exploration is likely to continue to delight, inform, and confound us for many years to come.