Role of cholesteryl ester transfer protein in selective uptake of high density lipoprotein cholesteryl esters by adipocytes

Previous reports attributed cholesteryl ester transfer protein (CETP)-mediated HDL cholesteryl ester (CE) selective uptake to the CETP-mediated transfer of CE from HDL to newly secreted apolipoprotein B-containing lipoproteins, which are then internalized by the LDL receptor (LDL-R). CETP has also been implicated in the remodeling of HDL, which renders it a better substrate for selective uptake by scavenger receptor class B type I (SR-BI). However, CETP-mediated selective uptake of HDL3-derived CE was not diminished in LDL-R null adipocytes, SR-BI null adipocytes, or in the presence of the receptor-associated protein. We found that monensin treatment or energy depletion of the SW872 liposarcoma cells with 2-deoxyglucose and NaN3 had no effect on CETP-mediated selective uptake, demonstrating that endocytosis is not required. This is supported by data indicating that CETP transfers CE into a compartment from which it can be extracted by unlabeled HDL. CETP could also mediate the selective uptake of HDL3-derived triacylglycerol (TG) and phospholipid (PL). The CETP-specific kinetics for TG and CE uptake were similar, and both reached saturation at approximately 5 microg/ml HDL. In contrast, CETP-specific PL uptake did not attain saturation at 5 microg/ml HDL and was approximately 6-fold greater than the uptake of CE. We propose two possible mechanisms to account for the role of CETP in selective uptake.     

Regulation of the selective uptake of high density lipoprotein-associated cholesteryl esters by human fibroblasts and Hep G2 hepatoma cells

We have previously shown that the liver and steroidogenic tissues of rats in vivo and a wider range of cells in vitro, including human cells, selectively take up high density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL particles. This process is regulated in tissues of rats and in cultured rat cells according to their cholesterol status. In the present study, we examined regulation of HDL selective uptake in cultured human fibroblasts and Hep G2 hepatoma cells. The cholesterol content of these cells was modified by a 20-hr incubation with either low density lipoprotein (LDL) or free cholesterol. Uptake of HDL components was examined in a subsequent 4-6-hr assay using intracellularly trapped tracers: 125I-labeled N-methyl-tyramine-cellobiose-apoA-I (125I-NMTC-apoA-I) to trace apoA-I, and [3H]cholesteryl oleyl ether to trace cholesteryl esters. In the case of fibroblasts, pretreatment with either LDL or free cholesterol resulted in decreased selective uptake (total [3H]cholesteryl ether uptake minus that due to particle uptake as measured by 125I-NMTC-apoA-I). In contrast, HDL particle uptake increased with either form of cholesterol loading. The amount of HDL that was reversibly cell-associated (bound) was increased by prior exposure to free cholesterol, but was decreased by prior exposure to LDL. In the case of Hep G2 cells, exposure to free cholesterol only slightly increased HDL particle uptake; selective uptake decreased after both forms of cholesterol loading, and reversibly bound HDL increased after exposure to free cholesterol, but either did not change or decreased after exposure to LDL. It was excluded that either LDL carried over into the HDL uptake assay or that products secreted by the cultured cells influenced these results. Thus, selective uptake by cells of both hepatic and extrahepatic origin was down-regulated by cholesterol loading, under which conditions HDL particle uptake increased. Total HDL binding was not directly correlated with either the rate of selective uptake or the rate of HDL particle uptake or the cholesterol status of the cells, suggesting more than one type of HDL binding site.     

Lipoprotein lipase enhances the cholesteryl ester transfer protein-mediated transfer of cholesteryl esters from high density lipoproteins to very low density lipoproteins

These studies were undertaken to examine the effects of lipoprotein lipase (LPL) and cholesteryl ester transfer protein (CETP) on the transfer of cholesteryl esters from high density lipoproteins (HDL) to very low density lipoproteins (VLDL). Human or rat VLDL was incubated with human HDL in the presence of either partially purified CETP, bovine milk LPL or CETP plus LPL. CETP stimulated both isotopic and mass transfer of cholesteryl esters from HDL into VLDL. LPL caused only slight stimulation of cholesteryl ester transfer. However, when CETP and LPL were both present, the transfer of cholesteryl esters from HDL into VLDL remnants was enhanced 2- to 8-fold, compared to the effects of CETP alone. The synergistic effects of CETP and LPL on cholesteryl ester transfer were more pronounced at higher VLDL/HDL ratios and increased with increasing amounts of CETP. In time course studies the stimulation of cholesteryl ester transfer activity occurred during active triglyceride hydrolysis. When lipolysis was inhibited by incubating LPL with either 1 M NaCl or 2 mM diethylparanitrophenyl phosphate, the synergism of CETP and LPL was reduced or abolished, and LPL alone did not stimulate cholesteryl ester transfer. These experiments show that LPL enhances the CETP-mediated transfer of cholesteryl esters from HDL to VLDL. This property of LPL is related to lipolysis.     

VLDL-bound lipoprotein lipase facilitates the cholesteryl ester transfer protein-mediated transfer of cholesteryl esters from HDL to VLDL

In recent years, it has been established that lipoprotein lipase (LPL) is partly associated with circulating lipoproteins. This report describes the effects of physiological amounts of very low density lipoprotein (VLDL)-bound LPL on the cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester transfer (CET) from high density lipoprotein (HDL) to VLDL. Three patients with severe LPL deficiency exhibited a strong decrease in net mass CET that was more than 80% lower than that of common hypertriglyceridemic subjects. Recombination experiments showed that this was due to an abnormal behavior of the VLDL fraction. Replacement of the latter by normal VLDL totally normalized net mass CET. We therefore prepared VLDL containing controlled amounts of bound LPL that we used as CE acceptors in experiments involving unidirectional radioisotopic CET measurements. These were carried out either in the absence or in the presence of inhibitors of LPL lipolytic activity. When LPL-induced lipolysis was totally blocked, the stimulating effect of the enzyme on the CETP-dependent CET was only reduced by about 50%, showing that it did not entirely result from its lipolytic action. These data were dependent upon neither the type of LPL inhibitor (E600 or THL) nor the source of CETP (delipidated plasma or partially purified CETP). Thus, in addition to the well-known stimulating effect of LPL-dependent lipolysis on CET, our work demonstrates that physiological amounts of VLDL-bound LPL may facilitate CET through a mechanism partially independent of its lipolytic activity.     

Comparative acyl specificities for transfer and selective uptake of high density lipoprotein cholesteryl esters

This study compares the specificities of selective uptake and transfer mediated by plasma cholesteryl ester transfer protein (CETP) for various species of cholesteryl esters in high density lipoproteins (HDL). [3H]Cholesterol was esterified with a series of variable chain length saturated acids and a series of variably unsaturated 18-carbon acids. These were incorporated into synthetic HDL particles along with 125I-labeled apoA-I as a tracer of HDL particles and [14C]cholesteryl oleate as an internal standard for normalization between preparations. Selective uptake by Y1-BS1 mouse adrenal cortical tumor cells was most extensively studied, but uptake by human HepG2 hepatoma cells and fibroblasts of human, rat, and rabbit origin were also examined. Acyl chain specificities for selective uptake and for CETP-mediated transfer were conversely related; selective uptake by all cell types decreased with increasing acyl chain length and increased with the extent of unsaturation of C18 chains. In contrast, CETP-mediated transfer increased with acyl chain length, and decreased with unsaturation of C18 chains. The specificities of human and rabbit CETP were also compared, and were found to differ little. Associated experiments showed that HDL-associated triglycerides, traced by [3H]glyceryl trioleyl ether, were selectively taken up but at a lesser rate than cholesteryl esters. The mechanism of this uptake appears to be the same as for selective uptake of cholesteryl esters.     

Rat adrenal uptake and metabolism of high density lipoprotein cholesteryl ester

Metabolism of high density lipoprotein (HDL) cholesteryl ester (CE) by cultured rat adrenal cells was studied. Addition of [3H]CE-HDL to cells pretreated with adrenocorticotrophin in lipoprotein poor media resulted in a time- and concentration-dependent accumulation of [3H]cholesteryl ester and production of [3H]cholesterol and [3H]corticosterone. HDL-CE metabolism could be described as the sum of a high affinity ([ HDL-cholesterol]1/2 max = 16 micrograms/ml) and low affinity ([ HDL-cholesterol]1/2 max greater than 70 micrograms/ml) process. [3H]Cholesterol was found both intracellularly and in the media. Accumulation of [3H]cholesteryl ester could not be attributed to uptake and re-esterification of unesterified cholesterol since addition of Sandoz 58-035, an inhibitor of acyl coenzyme A:cholesterol acyltransferase, did not prevent ester accumulation. Moreover, addition of chloroquine did not inhibit cholesteryl ester hydrolysis indicating that hydrolysis was not lysosomally mediated. Aminoglutethimide prevented conversion of [3H]CE-HDL to steroid hormones but did not inhibit [3H]cholesteryl ester uptake. Cellular accumulation of [3H] cholesteryl ester exceeded accumulation of 125I-apoproteins 5-fold at 1 h and 35-fold at 24 h indicating selective uptake of cholesteryl ester moiety. We conclude that rat adrenal cells possess a mechanism for selective uptake of HDL cholesteryl esters which provides substrate for steroidogenesis. These results constitute the first direct demonstration that cholesteryl esters in HDL can be used as steroidogenic substrate by the rat adrenal cortex.     

Human plasma cholesteryl ester transfer protein enhances the transfer of cholesteryl ester from high density lipoproteins into cultured HepG2 cells

The role of human plasma cholesteryl ester transfer protein (CETP) in the cellular uptake of high density lipoprotein (HDL) cholesteryl ester (CE) was studied in a liver tumor cell line (HepG2). When HepG2 cells were incubated with [3H]cholesteryl ester-labeled HDL3 in the presence of increasing concentrations of CETP there was a progressive increase in cell-associated radioactivity to levels that were 2.8 times control. The CETP-dependent uptake of HDL-CE was found to be saturated by increasing concentrations of both CETP and HDL. The CETP-dependent uptake of CE radioactivity increased continuously during an 18-h incubation. In contrast to the effect on cholesteryl ester, CETP failed to enhance HDL protein cell association or degradation. Enhanced uptake of HDL cholesteryl ester was shown for the d greater than 1.21 g/ml fraction of human plasma, partially purified CETP, and CETP purified to homogeneity, but not for the d greater than 1.21 g/ml fraction of rat plasma which lacks cholesteryl ester transfer activity. HDL cholesteryl ester entering the cell under the influence of CETP was largely degraded to free cholesterol by a process inhibitable by chloroquine. CETP enhanced uptake of HDL [3H]CE in cultured smooth muscle cells and to a lesser extent in fibroblasts but did not significantly influence uptake in endothelial cells or J774 macrophages. These experiments show that, in addition to its known role in enhancing the exchange of CE between lipoproteins, plasma CETP can facilitate the in vitro selective transfer of CE from HDL into certain cells.     

The study of nonspecific internalization of cholesteryl esters by the Hep G2 hepatoma cells

The cholesteryl oleate-POPC dispersions (1:3, mol/mol, mean particle size 110+/-20 nm) were taken up by the human hepatoma line Hep G2 cells via endocytosis. Internalization of the cholesteryl oleate-POPC dispersions by Hep G2 cells was dependent on the incubation time and dispersion concentration. At the cholesteryl oleate concentration 100 microM, its total uptake and internalization were found to be 1.5 nmol and 0.8 nmol per 1 mg of cell protein/24 h, respectively. Intracellular cleavage of the cholesteryl oleate incorporated in dispersions resulted in accumulation of free cholesterol capable of being released into the medium and metabolized to water-soluble polar products, presumably bile acids; oleic acid released is, apparently, involved in biosynthesis of triacylglycerides. The low-density lipoprotein receptor is not involved in internalization of lipid dispersions, and the presence of the cholesteryl oleate-POPC dispersions has no effect on the receptor-dependent internalization of cholesteryl esters of the low-density lipoproteins. The obtained data allow us to consider nonspecific internalization of cholesteryl esters by hepatocytes as a substantial part of the nonpolar lipid clearance.     

Transfer of cholesteryl ester into high density lipoprotein by cholesteryl ester transfer protein: effect of HDL lipid and apoprotein content

Recombinant high density lipoprotein (rHDL) particles were prepared by cosonication of purified lipids and human apoproteins and incubated with partly purified cholesteryl ester transfer protein (CETP) and low density lipoprotein (LDL) containing [3H]cholesteryl ester. Increasing the triglyceride content relative to cholesteryl ester in rHDL significantly decreased the ability of the particles to accept cholesteryl esters transferred by CETP. Kinetic analysis of the data was performed to numerically define the maximum velocity of lipid transfer, Tmax, and the HDL concentration required for half maximal velocity, KH. Increases in rHDL-triglyceride content were shown to result in a significant reduction in the Tmax without a major change in KH. When the free cholesterol content was increased relative to phospholipid, the ability of the particles to accept cholesteryl esters was also decreased in a similar manner. Conversely, rHDL prepared from purified apoprotein A-I, A-II, or mixtures of both, had significantly elevated Tmax and KH values for their interaction with CETP. The results suggest that increases in triglyceride or free cholesterol content of an rHDL particle decrease the catalytic ability of CETP by noncompetitive inhibition. In addition, some component(s) of HDL apoproteins, other than A-I or A-II, were shown to uncompetitively inhibit the activity of CETP, by modifying both Tmax and the KH for the reaction. This study has shown that altered HDL composition may have marked effects on the transfer and equilibration of cholesteryl esters within the HDL pool.     

Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice

The human cholesteryl ester transfer protein (CETP) facilitates the exchange of neutral lipids among lipoproteins. In order to evaluate the effects of increased plasma CETP on lipoprotein levels, a human CETP minigene was placed under the control of the mouse metallothionein-I promoter and used to develop transgenic mice. Integration of the human CETP transgene into the mouse genome resulted in the production of active plasma CETP. Zinc induction of CETP transgene expression caused depression of serum cholesterol due to a significant reduction of high density lipoprotein cholesterol. There was no change in total cholesterol content in very low and low density lipoproteins. However, there was a decrease in the free cholesterol/cholesteryl ester ratio in plasma and in all lipoprotein fractions of transgenic mouse plasma, suggesting stimulation of plasma cholesterol esterification. The results suggest that high levels of plasma CETP activity may be a cause of reduced high density lipoproteins in humans.     

Mechanism of scavenger receptor class B type I-mediated selective uptake of cholesteryl esters from high density lipoprotein to adrenal cells.

Despite extensive studies and characterizations of the high density lipoprotein-cholesteryl ester (HDL-CE)-selective uptake pathway, the mechanisms by which the hydrophobic CE molecules are transferred from the HDL particle to the plasma membrane have remained elusive, until the discovery that scavenger receptor BI (SR-BI) plays an important role. To elucidate the molecular mechanism, we examined the quantitative relationships between the binding of HDL and the selective uptake of its CE in the murine adrenal Y1-BS1 cell line. A comparison of concentration dependences shows that half-maximal high affinity cell association of HDL occurs at 8.7 +/- 4.7 micrograms/ml and the Km of HDL-CE-selective uptake is 4.5 +/- 1.5 micrograms/ml. These values are similar, and there is a very high correlation between these two processes (r2 = 0.98), suggesting that they are linked. An examination of lipid uptake from reconstituted HDL particles of defined composition and size shows that there is a non-stoichiometric uptake of HDL lipid components, with CE being preferred over the major HDL phospholipids, phosphatidylcholine and sphingomyelin. Comparison of the rates of selective uptake of different classes of phospholipid in this system gives the ranking: phosphatidylserine > phosphatidylcholine approximately phosphatidylinositol > sphingomyelin. The rate of CE-selective uptake from donor particles is proportional to the amount of CE initially present in the particles, suggesting a mechanism in which CE moves down its concentration gradient from HDL particles docked on SR-BI into the cell plasma membrane. The activation energy for CE uptake from either HDL3 or reconstituted HDL is about 9 kcal/mol, indicating that HDL-CE uptake occurs via a non-aqueous pathway. HDL binding to SR-BI allows access of CE molecules to a "channel" formed by the receptor from which water is excluded and along which HDL-CE molecules move down their concentration gradient into the cell plasma membrane.     

Low density lipoprotein uptake: holoparticle and cholesteryl ester selective uptake.

Low density lipoproteins (LDL) contain apolipoprotein B-100 and are cholesteryl ester-rich, triglyceride-poor macromolecules, arising from the lipolysis of very low density lipoproteins. This review will describe the receptors responsible for uptake of whole LDL particles (holoparticle uptake), and the selective uptake of LDL cholesteryl ester. The LDL-receptor mediates the internalization of whole LDL through an endosomal-lysosomal pathway, leading to complete degradation of LDL. Increasing LDL-receptor expression by pharmacological intervention efficiently reduces blood LDL concentrations. The lipolysis stimulated receptor and LDL-receptor related protein may also lead to complete degradation of LDL in presence of free fatty acids and apolipoprotein E- or lipase-LDL complexes, respectively. Selective uptake of LDL cholesteryl ester has been demonstrated in the liver, especially in rodents and humans. This activity brings five times more LDL cholesteryl ester than the LDL-receptor to human hepatoma cells, suggesting that it is a physiologically significant pathway. The lipoprotein binding site of HepG2 cells mediates this process and recognizes all lipoprotein classes. Scavenger receptor class B type I and CD36, which mediate the selective uptake of high density lipoprotein cholesteryl ester, are potentially involved in LDL cholesteryl ester selective uptake, since they both bind LDL with high affinity. It is not known whether they are identical to the uncloned lipoprotein binding site and if the selective uptake of LDL cholesteryl ester produces a less atherogenic particle. If this is verified, pharmacological up-regulation of LDL cholesteryl ester selective uptake may become another therapeutic approach for reducing blood LDL-cholesterol levels and the risk of atherosclerosis.     

The low density lipoprotein receptor-related protein contributes to selective uptake of high density lipoprotein cholesteryl esters by SW872 liposarcoma cells and primary human adipocytes

The concept that selective transfer of high density lipoprotein (HDL)-derived cholesteryl esters (CE) does not require lipoprotein internalization has been challenged recently by evidence that implicates HDL recycling during the selective uptake process. This has prompted us to examine the role of the low density lipoprotein receptor-related protein (LRP) in selective uptake. LRP is an endocytic receptor for lipoprotein lipase (LpL) and apolipoprotein E (apoE) ligands that are able to mediate selective uptake. We report that molecules that interfere with ligand binding to LRP, such as the receptor-associated protein (RAP), suramin, alpha(2)-macroglobulin, or lactoferrin, inhibit HDL-CE selective uptake by human primary adipocytes and SW872 liposarcoma cells by 35-50%. This partial inhibition of selective uptake from total HDL was not due to preferential inhibition of the HDL(2) or HDL(3) subfractions. Selective uptake by the scavenger receptor BI was not inhibited by RAP, excluding its involvement. Furthermore, in SW872 cells in which LRP was reduced to 14% of control levels by stable antisense expression, selective uptake was attenuated by at least 33%, confirming a role for LRP in this process. RAP, alpha(2)-macroglobulin, lactoferrin, and suramin (individually or in paired combinations) also attenuated selective uptake of HDL-CE by primary human adipocytes by about 40%. On the other hand, human skin fibroblasts express LRP abundantly but lack the capacity for selective uptake, demonstrating that other molecules are required. In SW872 cells, exogenous apoE or LpL can facilitate selective uptake but only the apoE-enhanced uptake can be inhibited by RAP, implicating apoE as a likely co-mediator. We discuss the possible mechanisms by which the endocytic receptor, LRP, can mediate selective uptake.     

Inhibition of cholesteryl ester transfer protein by anacetrapib does not impair the anti-inflammatory properties of high density lipoprotein

Cholesteryl ester transfer protein (CETP) is a target of therapeutic intervention for coronary heart disease. Anacetrapib, a potent inhibitor of CETP, has been shown to reduce LDL-cholesterol by 40% and increase HDL-cholesterol by 140% in patients, and is currently being evaluated in a phase III cardiovascular outcomes trial. HDL is known to possess anti-inflammatory properties, however with such large increases in HDL-cholesterol, it is unclear whether CETP inhibition perturbs HDL functionality such as anti-inflammatory effects on endothelial cells. The purpose of the present study was to determine whether CETP inhibition by anacetrapib affects the anti-inflammatory properties of HDL. HDL was isolated from either hamsters treated with vehicle or anacetrapib for 2 weeks, or from normal human subjects treated either placebo, 20 mg, or 150 mg anacetrapib daily for 2 weeks. Anacetrapib treatment increased plasma HDL cholesterol levels by 65% and between 48 and 82% in hamsters and humans, respectively. Pre-incubation of human aortic endothelial cells with HDL isolated from both control and anacetrapib treated hamsters suppressed TNFα induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin. Similar results were obtained with human HDL samples pre and post treatment with placebo or anacetrapib. Further, HDL inhibited TNFα-induced MCP-1 secretion, monocyte adhesion and NF-κB activation in endothelial cells, and the inhibition was similar between control and anacetrapib treated groups. These studies demonstrate that anacetrapib treatment does not impair the ability of HDL to suppress an inflammatory response in endothelial cells.     

Regulation of the selective uptake of high density lipoprotein-associated cholesteryl esters

We have previously shown in rats that the cholesteryl ester component of high density lipoproteins (HDL) is taken up at a greater fractional rate than is the apolipoprotein A-I component (selective uptake) by liver and steroidogenic tissues. Selective uptake was also exhibited by cultured cells from these organs as well as by a wider range of cells in vitro (e.g., rat and human fibroblasts). We report here regulation of this pathway according to the cholesterol status of cells. Uptake of HDL cholesteryl esters by rat fibroblasts was decreased by prior loading of the cells with cholesterol, even while uptake of HDL-associated apoA-I actually increased. At high levels of cholesterol, the two were taken up about in parallel, i.e., selective uptake was suppressed. A similar regulation of selective uptake in primary rat hepatocytes in culture was not observed. To examine regulation of selective uptake in vivo, hypocholesterolemia was induced in rats using either 4-aminopyrazolo[3,4-d]pyrimidine or 17 alpha-ethinyl estradiol. Rat HDL, doubly labeled in both the apoprotein A-I and cholesteryl ester moieties with intracellularly trapped tracers, were injected into untreated and treated rats. The plasma decay kinetics and the tissue sites of uptake were then determined. Hypocholesterolemia increased the plasma fractional catabolic rates of both tracers. Selective uptake was observed in tissues of treated rats that did not exhibit selective uptake in untreated rats (muscle, adipose tissue, and skin). Similarly, hypocholesterolemia increased the contribution of selective uptake to total HDL cholesteryl ester uptake by adrenal and ovary. In contrast, regulation of selective uptake by liver could not be demonstrated under these conditions. Thus, selective uptake of HDL cholesteryl esters can be regulated in extrahepatic tissues of rats in vivo and in vitro, suggesting a role for selective uptake in the maintenance of cholesterol homeostasis in these tissues.     

Plasma phospholipid transfer protein-mediated reactions are impaired by hypochlorite-modification of high density lipoprotein

The two main functions of phospholipid transfer protein (PLTP) are the transfer of phospholipids between plasma lipoproteins and the conversion of high density lipoprotein (HDL), where prebeta-HDL particles are generated. HDL is considered an anti-atherogenic lipoprotein due to its function in the reverse cholesterol transport, where prebeta-HDL accepts cellular membrane cholesterol from peripheral tissues. However, the anti-atherogenic properties of native HDL may be abolished by oxidation/modification. Hypochlorous acid/hypochlorite (HOCl/OCl-)-a potent oxidant generated in vivo only by the myeloperoxidase-H2O2-chloride system of activated phagocytes-alters the physiological properties of HDL by generating a pro-atherogenic lipoprotein particle. Therefore, we have studied the effect of HOCl on the function of HDL subclass 3 (HDL3) and triglyceride-enriched HDL3 (TG-HDL3) in PLTP-mediated processes in vitro. Modification of HDL3 and TG-HDL3 with increasing HOCl concentrations (oxidant:lipoprotein molar ratio between 25:1 and 200:1) decreased the capacity of the corresponding lipoprotein particles to accept phospholipids. Although binding of PLTP to unmodified and HOCl-modified lipoprotein particles was similar, the degree of PLTP-mediated HDL conversion was decreased upon HOCl oxidation. PLTP released apolipoprotein A-I (apoA-I) from HOCl-modified HDL3, but the particles formed displayed no prebeta-mobility. Based on these findings, we conclude that the substrate properties of HOCl-modified HDL3 and TG-HDL3 in PLTP-mediated processes are impaired, which indicates that the anti-atherogenic properties of HDL are impaired.     

Regulation of the selective uptake of cholesteryl esters from high density lipoproteins by sphingomyelin

Although sphingomyelin (SM) is a major phospholipid in lipoproteins as well as in the membrane rafts where the scavenger receptor class B type I (SR-BI) is localized, its possible role in the selective uptake of cholesteryl ester (CE) by the SR-BI-mediated pathway is unknown. We investigated the effect of SM in lipoproteins and cell membranes on the selective uptake in three different cell lines: SR-BI-transfected CHO cells, hepatocytes (HepG2), and adrenocortical cells (Y1BS1). Incorporation of SM into recombinant high density lipoprotein (rHDL) containing labeled CE resulted in up to 50% inhibition of the selective uptake of CE in all three cell lines. This inhibition was completely reversed by treatment of rHDL with sphingomyelinase (SMase). Selective uptake from plasma HDL was activated by 22-72% after treatment of HDL with SMase. In addition, pretreatment of the cells with SMase resulted in stimulation of CE uptake from rHDL by CHO and Y1BS1, although not by HepG2. Incorporation of ceramide into rHDL resulted in up to 2-fold stimulation of CE uptake, although pretreatment of cells with egg ceramide had no significant effect. These results show that SM and ceramide in the lipoproteins and the cell membranes regulate the SR-BI-mediated selective uptake of CE, possibly by interacting with the sterol ring or with SR-BI itself.     

Riboflavin uptake by the human-derived liver cells Hep G2: Mechanism and regulation

The water-soluble vitamin riboflavin (RF) plays a critical role in many metabolic reactions, and thus, is essential for normal cellular functions and growth. The liver plays a central role in normal RF metabolism and is the site of maximal utilization of the vitamin. The mechanism of liver uptake of RF has been studied in animals, but no information is available describing the mechanism of the vitamin uptake in the human situation and its cellular regulation. In this study, we used the human-derived liver cells Hep G2 as an in vitro model system to address these issues. Uptake of RF by Hep G2 cells was found to be temperature- and energy-dependent but Na⁺-independent in nature. Uptake seemed to involve a carrier-mediated process as indicated by the saturation as a function of substrate concentration (apparent K_m 0.41 ± 0.08 μM), and by the ability of the structural analogs lumiflavin and lumichrome to inhibit the uptake process [inhibition constant (K_i) of 1.84 and 6.32 μM, respectively]. RF uptake was energy dependent, and was inhibited by the -SH group blocker p-chloromercuriphenylsulfonate (p-CMPS) (K_i of 0.10 mM). Specific modulators of intracellular protein kinase A (PKA)-, protein kinase C (PKC)-, and protein tyrosine kinase (PTK)-mediated pathways did not affect RF uptake by Hep G2 cells. On the other hand, specific inhibitors of Ca²⁺/calmodulin-mediated pathway significantly inhibited the uptake process; this effect seemed to be mediated through a decrease in the V_max of the substrate uptake process. Maintaining Hep G2 cells in a RF-deficient growth medium was associated with a significant up-regulation in the substrate uptake; this effect was specific for RF and was mediated mainly by means of an increase in the V_max of the uptake process. These results describe, for the first time, the mechanism and cellular regulation of RF uptake by a human-derived liver cellular preparation, and shows the involvement of a carrier-mediated system in the uptake process. Furthermore, the uptake process seems to be regulated by an intracellular Ca²⁺/calmodulin-mediated pathway and by extracellular substrate levels. J. Cell. Physiol. 176:588–594, 1998. © 1998 Wiley-Liss, Inc.     

Genetic variations in the cholesteryl ester transfer protein gene and high density lipoprotein cholesterol levels in Taiwanese Chinese

This study analyzed the association of the I14A mutation, the D442G mutation, and the TaqIB polymorphism of the cholesteryl ester transfer protein (CETP) gene in 718 Chinese individuals with high-density lipoprotein cholesterol levels (HDL-C) living in Taiwan. The analysis revealed that the I14A mutation was not present in any of the 110 subjects with HDL-C levels above 60 mg/dl. By contrast, the D442G mutation was present in 48 of the 718 (6.7%) subjects tested. Significantly higher HDL-C levels were noted for bearers of the D442G mutation compared with non-bearers; however, this association was weaker for males and for subjects carrying the TaqIB1 allele. The TaqIB2 allele was also associated with higher HDL-C levels. From multivariate analysis, independent associations were demonstrated for the TaqIB2 polymorphism and the D442G mutation, and elevated HDL-C levels. For obese subjects, however, the presence of the TaqIB2 or D442G allele was not associated with increased HDL-C levels. For subjects with triglycerides at a concentration greater than 150 mg/dl, the association of both alleles with HDL-C levels was also diminished. Thus, genetic variation at the CETP gene locus may account for a significant proportion of the difference in HDL-C levels; however, it seems reasonable to suggest that the effects of the allele interact with genetic variations expressed within the sample population, and with sex, obesity, and plasma triglyceride levels.     

Regulation of the uptake of high-density lipoprotein-originated cholesteryl ester by HepG2 cells: role of low-density lipoprotein and plasma lipid transfer protein.

Cholesteryl ester uptake by the human hepatoma cell line HepG2 was studied in vitro by using radiolabeled cholesteryl ester as a tracer. After the cells were incubated in a lipoprotein deficient condition, the rate of radio labeled cholesteryl ester uptake from low-density lipoprotein (LDL) was estimated to be some 25-times higher than that from high-density lipoprotein (HDL). LDL-cholesteryl ester uptake was suppressed by preincubation of the cells with LDL, but pretreatment of the cells with HDL did not show significant effect. HDL-cholesteryl ester uptake was only slightly suppressed by pretreatment of the cells with LDL, and there was no effect with HDL pretreatment. HDL-cholesteryl ester uptake was not affected either by the presence of LDL or human plasma lipid transfer protein alone in the medium under our experimental conditions. Lipid transfer protein enhanced the uptake of radiolabeled cholesteryl ester originating from HDL by the cells only in the presence of LDL. Thus, lipid transfer protein catalyzes a bypass to LDL for the uptake by HepG2 cells of cholesteryl ester molecules which originate in HDL, and this pathway is much more efficient than direct uptake of cholesteryl ester originating in HDL by these cells.