The selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE) by the scavenger receptor class B type I (SR-BI) is well documented. However, the effect of altered HDL composition, such as occurs in hyperlipidemia, on this important process is not known. This study investigated the impact of variable CE and triglyceride (TG) content on selective uptake. CE selective uptake by Y1 and HepG2 cells was strongly affected by modification of either the CE or TG content of HDL. Importantly, TG, like CE, was selectively taken up by a dose-dependent, saturable process in these cells. As shown by ACTH up-regulation and receptor overexpression experiments, SR-BI mediated the selective uptake of both CE and TG. With in vitro modified HDLs of varying CE and TG composition, the selective uptake of CE and TG was dependent on the abundance of each lipid within the HDL particle. Furthermore, total selective uptake (CE ؉ TG) remained constant, indicating that these lipids competed for cellular uptake. These data support a novel mechanism whereby SR-BI binds HDL and mediates the incorporation of a nonspecific portion of the HDL lipid core. In this way, TG directly affects the ability of HDL to donate CE to cells. Processes that raise the TG/CE ratio of HDL will impair the delivery of CE to cells via this receptor and may compromise the efficiency of sterol balancing pathways such as reverse cholesterol transport.

Expression of the adenovirus protein RIDα rescues the cholesterol storage phenotype in NPC1-deficient cells by inducing formation of lipid droplets. The function of RIDα is independent of NPC1 but dependent on NPC2 and the oxysterol-binding protein ORP1L. This study provides the first evidence that ORP1L plays a role in sterol transport and LD formation.

Lipid transfer inhibitor protein (LTIP) is an important regulator of cholesteryl ester transfer protein function. We report the development of an immunoassay for LTIP and its use to quantify LTIP in plasma of varying lipid contents. A rabbit antibody against bacterially produced recombinant LTIP detected two LTIP isoforms in plasma differing in carbohydrate content. This antibody was used in a competitive, enzyme-linked immunoassay that uses partially purified LTIP bound to microtiter plates. To optimize LTIP immunoreactivity, plasma samples required preincubation in 1% Tween-20 and 0.5% Nonidet P-40. In normolipidemic plasma, LTIP averaged 83.5 mg/ml. LTIP was 31% higher in males than in females. LTIP was positively associated with HDL cholesterol in normolipidemic males but not in females. In hypertriglyceridemic males, LTIP was only 56% of control values, whereas in hypertriglyceridemic females, LTIP tended to increase. Additionally, in males with normal cholesterol and triglyceride (TG) < 200 mg/dl, LTIP varied inversely with plasma TG. Overall, we have confirmed the negative association between plasma TG levels and LTIP previously suggested by a small data set, but now we demonstrate that this effect is seen only in males. The mechanisms underlying this gender-specific response to TG, and why LTIP and HDL levels correlate in males but not in females, remain to be determined. In plasma, cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester (CE) from LDL and HDL to VLDL in return for triglyceride (TG) (1, 2). Several lines of evidence suggest that lipid transfer inhibitor protein (LTIP), also known as apolipoprotein F (3), alters this process and influences the capacity of CETP to remodel lipoproteins. We have shown that among normolipidemic subjects, LTIP activity levels in whole plasma associate negatively with the rate of lipid transfer between VLDL and LDL (4). The addition of exogenous LTIP to native plasma reduces the participation of LDL in lipid transfer events but leads to a dose-dependent increase in the efflux of CE from HDL to VLDL, resulting in HDLs that are markedly better substrates for lecithin:cholesterol acyltransferase (5). We have proposed that this increased CETP activity on HDL happens because plasma TG, not CETP, is rate-limiting for net lipid transfer in normal plasma (6, 7). Thus, in the presence of LTIP, less VLDL TG is expended by transfer to LDL, allowing the transfer from VLDL to HDL to be increased.We substantiated the foregoing observations through studies of patients undergoing continuous ambulatory peritoneal dialysis, who have very low LTIP activity (8). In these patients, the 2-fold preference of CETP for HDL as a lipid donor (compared with LDL) seen in normal plasma is absent. In separate studies, we determined that CETP actually displays no preference for total HDL as a substrate in assays with isolated lipoproteins; however, the addition of plasma levels of LTIP to these assays completely reconstitutes the 2-fold preference of C...

Lipid transfer inhibitor protein (LTIP) is a physiologic regulator of cholesteryl ester transfer protein (CETP) function. We previously reported that LTIP activity is localized to LDL, consistent with its greater inhibitory activity on this lipoprotein. With a recently described immunoassay for LTIP, we investigated whether LTIP mass is similarly distributed. Plasma fractionated by gel filtration chromatography revealed two LTIP protein peaks, one coeluting with LDL, and another of ?470 kDa. The 470 kDa LTIP complex had a density of 1.134 g/ml, indicating ?50% lipid content, and contained apolipoprotein A-I. By mass spectrometry, partially purified 470 kDa LTIP also contains apolipoproteins C-II, D, E, J, and paraoxonase 1. Unlike LDL-associated LTIP, the 470 kDa LTIP complex does not inhibit CETP activity. In normolipidemic subjects, ?25% of LTIP is in the LDLassociated, active form. In hypercholesterolemia, this increases to 50%, suggesting that lipoprotein composition may influence the status of LTIP activity. Incubation (37°C) of normolipidemic plasma increased active, LDL-associated LTIP up to 3-fold at the expense of the inactive pool. Paraoxon inhibited this shift by 50%. Overall, these studies show that LTIP activity is controlled by its reversible incorporation into an inactive complex. This may provide for short-term fine-tuning of lipoprotein remodeling mediated by CETP.-He, Y., D. J. Greene, M. Kinter, and R. E. Morton. In plasma, cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester (CE) from LDL and HDL to VLDL in return for triglyceride (TG) (1, 2). This remodeling of lipoprotein composition alters the metabolism of lipoproteins and ultimately influences both the quality and quantity of lipoproteins in plasma (3-5). Physiologically, CETP may be regulated by at least two proteins. Apolipoprotein C-I, which resides primarily on HDL, has been reported to inhibit CETP in vitro, and studies with transgenic animals have demonstrated its ability to suppress CETP activity in vivo (6, 7). Its mode of action is via modification of the surface charge of HDL, resulting in weakened CETP-HDL interactions and thus suppression of lipid transfer events with HDL (8). A second regulatory protein is lipid transfer inhibitor protein (LTIP), also known as apolipoprotein F (9). Although the mechanism of inhibition of CETP by LTIP has not been firmly established, it also appears to function by disrupting the interaction of CETP with its substrate (10). However, the effects of LTIP on lipid transfer events are quite distinct from that of apolipoprotein C-I. Unlike apolipoprotein C-I, LTIP activity is associated with LDL (11). The addition of exogenous LTIP to native plasma reduces the participation of LDL in lipid transfer events but leads to a dose-dependent increase in the efflux of CE from HDL to VLDL. This enhanced CETP activity on HDL results in HDL particles that are markedly better substrates for lecithin:cholesterol acyltransferase (11). We have proposed that this increa...

The multidrug resistance P-glycoprotein (P-gp) was recently proposed to redistribute cholesterol in the plasma membrane, suggesting that P-gp could modulate cholesterol efflux to cholesterol acceptors. To address this hypothesis and to reevaluate the role of P-gp in cholesterol homeostasis, we first analyzed the role of P-gp expression on cholesterol efflux in P-gp stably transfected drug-selected LLC-MDR1 cells. Cholesterol efflux to methyl-b-cyclodextrin (CD) was 4-fold higher in LLC-MDR1 cells compared with control LLC-PK1 cells, indicating that the accessible pool of plasma membrane cholesterol was increased by P-gp expression. However, using the P-gp-inducible cells lines HeLa MDR-Tet and 77.1 MDR-Tet, cholesterol efflux to CD, apolipoprotein A-I, or HDL was not associated with P-gp expression. In addition, we did not observe any effect of Pgp expression on cellular free and esterified cholesterol content, cholesteryl ester uptake from LDL and HDL particles, or acyl-CoA:cholesterol acyltransferase activity. Therefore, we conclude that P-gp expression does not play a major role in cholesterol homeostasis in P-gp-inducible cells and that the effects of P-gp on cholesterol homeostasis previously described in drug-selected cells might result from non-P-gp pathways that were also induced by selection for drug resistance.-Le Goff, W., M. Settle, D. J. Greene, R. E. Morton, and J. D. Smith. Reevaluation of the role of the multidrug-resistant P-glycoprotein in cellular cholesterol homeostasis. J. Lipid Res. 2006. 47: 51-58. Supplementary key words cholesterol efflux . ABCA1 . cyclodextrin . apolipoprotein A-I P-glycoprotein (P-gp) is a member of the ATP binding cassette transporter family responsible for the multidrug resistance (MDR) phenotype. P-gp is the protein product of the human MDR1 gene (ABCB1 is the official gene symbol), and P-gp is highly expressed in the intestine, liver, kidney, placenta, and the blood-brain barrier. Plasma membrane P-gp mediates the efflux of numerous neutral and cationic organic compounds and drugs, among them chemotherapeutic drugs, thereby contributing to the MDR phenotype in many cancers. However, information concerning endogenous substrates for P-gp as well as the physiological role of P-gp is still lacking (1).P-gp has been reported to modulate cellular cholesterol homeostasis via several mechanisms. Stable transfection of a rat intestinal cell line with a P-gp expression vector led to a modest increase in the uptake of cholesterol-containing micelles (2). Nonspecific P-gp inhibitors have been reported to inhibit cholesterol biosynthesis in CHO-7 cells (3) and to inhibit cellular cholesterol esterification (4). Transfection of NIH 3T3 cells with a P-gp expression vector followed by selection for drug resistance was associated with increased esterification of plasma membrane cholesterol (5). Mice, unlike humans, have two copies of the gene for P-gp, abcb1a and abcb1b (previously mdr1a and mdr1b). Mice deficient in both of these genes have been constructed, and althou...