Remodeling and shuttling - Mechanisms for the synergistic effects between different acceptor particles in the mobilization of cellular cholesterol

In normal physiology, cells are exposed to cholesterol acceptors of different sizes simultaneously. The current study examined the possible interactions between two different classes of accepters, one large (large unilamellar phospholipid vesicles, LUVs) and one small (HDL or other small accepters), added separately or in combination to Fu5AH rat hepatoma cells. During a 24-hour incubation, LUVs of palmitoyl-oleoyl phosphatidylcholine at 1 mg phospholipid (PL) per milliliter extracted approximate to 20% of cellular unesterified cholesterol (UC) label and mass in a slow, continuous fashion (half-time [t 1/2] for UC efflux was approximate to 50 hours) and human HDL(3) at 25 mu g PL per milliliter extracted approximate to 15% cellular UC label with no change in cellular cholesterol mass (t 1/2 of approximate to 8 hours). In contrast, the combination of LUVs and HDL(3) extracted over 90% of UC label (t 1/2 of approximate to 4 hours) and approximate to 50% of the UC mass, indicating synergy. To explain this synergy, specific particle interactions were examined, namely, remodeling, in which the two accepters alter each other's composition and thus the ability to mobilize cellular cholesterol, and shuttling, in which the small acceptor ferries cholesterol from cells to the large acceptor. To examine remodeling, LUVs and HDL were coincubated and reisolated before application to cells. This HDL became UC depleted, PL enriched, and lost a small amount of apolipoprotein A-I, Compared with equivalent numbers of control HDL particles, remodeled HDL caused faster efflux (t 1/2 approximate to 4 hours) and exhibited a greater capacity to sequester cellular cholesterol over 24 hours (approximate to 38% versus approximate to 15% for control HDL), consistent with their enrichment in PL. Remodeled LUVs still extracted approximate to 20% of cellular UC. Thus, remodeling accounted for some but not all of the synergy between LUVs and HDL. To examine shuttling, several approaches were used. First, reisolation of particles after an 8-hour exposure to cells revealed that HDL contained very little of the cellular UC label. The label was found almost entirely with the LUVs, suggesting that LUVs continuously stripped the HDL of cellular UC. Second, bidirectional flux studies demonstrated that LUVs blocked the influx of HDL UC label into cells, while the rate of efflux of cellular UC was maintained. These kinetic effects explained the massive net loss of cellular UC to LUVs with HDL. Third, cyclodextrin, an artificial small acceptor that does not acquire PL and hence does not become remodeled, exhibited substantial synergy with LUVs, supporting shuttling. Thus, the presence of large and small accepters together can overcome intrinsic deficiencies in each. Small accepters are efficient at extracting cellular cholesterol because they approach cell surfaces easily but have a low capacity, whereas large accepters are inefficient but have a high capacity. When present simultaneously, where the small acceptor can transfer cholesterol quickly to the large acceptor, high efficiency and high capacity are achieved. The processes responsible for this synergy, namely, remodeling and shuttling, may be general phenomena allowing cooperation both during normal physiology and after therapeutic administration of accepters to accelerate tissue cholesterol efflux in vivo.