Cell surface adenylate kinase activity regulates the F(1)-ATPase/P2Y (13)-mediated HDL endocytosis pathway on human hepatocytes.

We have previously demonstrated on human hepatocytes that apolipoprotein A-I binding to an ecto-F(1)-ATPase stimulates the production of extracellular ADP that activates a P2Y(13)-mediated high-density lipoprotein (HDL) endocytosis pathway. Therefore, we investigated the mechanisms controlling the extracellular ATP/ADP level in hepatic cell lines and primary cultures to determine their impact on HDL endocytosis. Here we show that addition of ADP to the cell culture medium induced extracellular ATP production that was due to adenylate kinase [see text] and nucleoside diphosphokinase [see text] activities, but not to ATP synthase activity. We further observed that in vitro modulation of both ecto-NDPK and AK activities could regulate the ADP-dependent HDL endocytosis. But interestingly, only AK appeared to naturally participate in the pathway by consuming the ADP generated by the ecto-F(1)-ATPase. Thus controlling the extracellular ADP level is a potential target for reverse cholesterol transport regulation.

The nucleotide receptor P2Y13 is a key regulator of hepatic high-density lipoprotein (HDL) endocytosis.

Cell surface receptors for high-density lipoprotein (HDL) on hepatocytes are major partners in the regulation of cholesterol homeostasis. We recently identified a cell surface ATP synthase as a high-affinity receptor for HDL apolipoprotein A-I (apoA-I) on human hepatocytes. Stimulation of this ectopic ATP synthase by apoA-I triggered a low-affinity-receptor-dependent HDL endocytosis by a mechanism strictly related to the generation of ADP. This suggests that nucleotide G-protein-coupled receptors of the P2Y family are molecular components in this pathway. Only P2Y1 and P2Y13 are present on the membrane of hepatocytes. Using both a pharmacological approach and small interference RNA, we identified P2Y13 as the main partner in hepatic HDL endocytosis, in cultured cells as well as in situ in perfused mouse livers. We also found a new important action of the antithrombotic agent AR-C69931MX as a strong activator of P2Y13-mediated HDL endocytosis.

New insight on the molecular mechanisms of high-density lipoprotein cellular interactions.

High-density lipoprotein (HDL) cholesterol is an independent negative risk factor for coronary artery disease and thus represents today the only protective factor against atherosclerosis. The protective effect of HDL is mostly attributed to its central function in reverse cholesterol transport (RCT), a process whereby excess cell cholesterol is taken up and processed in HDL particles, and is later delivered to the liver for further metabolism and bile excretion. This process relies on specific interactions between HDL particles and cells, both peripheral (cholesterol efflux) and hepatic (cholesterol disposal) cells, and on the maturation of HDL particles within the vascular compartment. The plasma level of HDL cholesterol will thus result also from the complex interplay with cellular partners. Among them, some contribute to HDL formation - for instance ATP binding cassette AI protein - while others are mostly involved in HDL catabolism, the scavenger receptor-class B type I or the recently described membrane-bound ATP synthase/hydrolase. The last decade has seen major breakthroughs in the identification and elucidation of the role of cellular partners of HDL metabolism, and in their transcriptional regulations, opening up new perspectives in the modulation of HDL cholesterol.

RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: evidenced in vivo by an anti-farnesyl cysteine antibody.

Protein isoprenylation is a lipid posttranslational modification required for the function of many proteins that share a carboxyl-terminal CAAX motif. The X residue determines which isoprenoid will be added to the cysteine. When X is a methionine or serine, the farnesyl-transferase transfers a farnesyl, and when X is a leucine or isoleucine, the geranygeranyl-transferase I, a geranylgeranyl group. But despite its CKVL motif, RhoB was reported to be both geranylgeranylated and farnesylated. Thus, the determinants of RhoB prenylation appear more complex than initially thought. To determine the role of RhoB CAAX motif, we designed RhoB mutants with modified CAAX sequence expressed in baculovirus-infected insect cells. We demonstrated that RhoB was prenylated as a function of the three terminal amino acids, i.e., RhoB bearing the CAIM motif of lamin B or CLLL motif of Rap1A was farnesylated or geranylgeranylated, respectively. Next, we produced a specific polyclonal antibody against farnesyl cysteine methyl ester allowing prenylation analysis avoiding the metabolic labeling restrictions. We confirmed that the unique modification of the RhoB CAAX box was sufficient to direct the RhoB distinct prenylation in mammalian cells and, inversely, that a RhoA-CKVL chimera could be alternatively prenylated. Moreover, the immunoprecipitation of endogenous RhoB from cells with the anti-farnesyl cysteine antibody suggested that wild-type RhoB is farnesylated in vivo. Taken together, our results demonstrated that the three last carboxyl amino acids are the main determinants for RhoB prenylation and described an anti-farnesyl cysteine antibody as a useful tool for understanding the cellular control of protein farnesylation.

Characterization of two high-density lipoprotein binding sites on porcine hepatocyte plasma membranes: contribution of scavenger receptor class B type I (SR-BI) to the low-affinity component.

Two HDL(3) high- and low-affinity binding sites are present on the human hepatoma cell line (HepG(2)). Recently, we have suggested that the high-affinity binding sites might modulate the endocytosis of HDL through the low-affinity binding sites [Guendouzi, K. (1998) Biochemistry 37, 14974-14980], highlighting the physiological importance of this family of HDL high-affinity binding sites. The present data demonstrate the presence of HDL(3) high-affinity (K(d) = 0.37 microg/mL, B(max) = 260 ng/mg of protein) and low-affinity (K(d) = 86.2 microg/mL, B(max) = 14 300 ng/mg of protein) binding sites on purified porcine hepatocyte plasma membranes. By contrast, free apoA-I was strictly specific to the high-affinity sites (K(d) = 0.2 microg/mL and B(max) = 72 ng/mg of protein). Competition experiments between (125)I-labeled HDL(3) and either LDL, oxidized LDL, or anti-SR-BI IgG as competitors show that SR-BI is mostly responsible (70% displacement) for the binding of HDL(3) to the low-affinity binding sites. By contrast, the same competition experiments using (125)I-labeled free apoA-I clearly excluded SR-BI as the high-affinity binding receptor. We conclude that the binding of HDL onto hepatocyte plasma membranes involves: (1) two low-affinity binding receptors, one being SR-BI; (2) one family of high-affinity binding sites unrelated to SR-BI.

Identification of an ApoA-I ligand domain that interacts with high-affinity binding sites on HepG2 cells.

We have previously described the presence of two (high- and low-affinity) HDL binding sites on the hepatoma cell line (HepG2) (R. Barbaras, X. Collet, H. Chap, and B. Perret (1994) Biochemistry 33, 2335-2340]. Moreover, apoA-I, the major HDL apolipoprotein, interacts with these two binding sites, while lipid-free apoA-I binds only to the high-affinity sites. Using tryptic HDL fragments and HepG2 cell monolayers as an "affinity matrix," we identified an apoA-I peptide of 16 amino acids, spanning between residues 62 and 77, as a ligand domain. The corresponding synthetic peptide displays high-affinity (K(d) approximately 10(-7) M) and low-capacity (B(max) 8 pmol/mg of cell protein) binding components. Competition experiments with this peptide, using (125)I-labeled free apoA-I as a ligand, show that this binding corresponds to the high-affinity binding sites already described. In conclusion, we identified the apoA-I 62-77 region as a specific high-affinity ligand domain of HDL on HepG2 cells.

Remodeling of HDL by CETP in vivo and by CETP and hepatic lipase in vitro results in enhanced uptake of HDL CE by cells expressing scavenger receptor B-I.

The transport of HDL cholesteryl esters (CE) from plasma to the liver involves a direct uptake pathway, mediated by hepatic scavenger receptor B-I (SR-BI), and an indirect pathway, involving the exchange of HDL CE for triglycerides (TG) of TG-rich lipoproteins by cholesteryl ester transfer protein (CETP). We carried out HDL CE turnover studies in mice expressing human CETP and/or human lecithin:cholesterol acyltransferase (LCAT) transgenes on a background of human apoA-I expression. The fractional clearance of HDL CE by the liver was delayed by LCAT transgene, while the CETP transgene increased it. However, there was no incremental transfer of HDL CE radioactivity to the TG-rich lipoprotein fraction in mice expressing CETP, suggesting increased direct removal of HDL CE in the liver. To evaluate the possibility that this might be mediated by SR-BI, HDL isolated from plasma of the different groups of transgenic mice was incubated with SR-BI transfected or control CHO cells. HDL isolated from mice expressing CETP showed a 2- to 4-fold increase in SR-BI-mediated HDL CE uptake, compared to HDL from mice lacking CETP. The addition of pure CETP to HDL in cell culture did not lead to increased selective uptake of HDL CE by cells. However, when human HDL was enriched with TG by incubation with TG-rich lipoproteins in the presence of CETP, then treated with hepatic lipase, there was a significant enhancement of HDL CE uptake. Thus, the remodeling of human HDL by CETP, involving CE;-TG interchange, followed by the action of hepatic lipase (HL), leads to the enhanced uptake of HDL CE by cellular SR-BI. These observations suggest that in animals such as humans in which both the selective uptake and CETP pathways are active, the two pathways could operate in a synergistic fashion to enhance reverse cholesterol transport.

Biochemical and physical properties of remnant-HDL2 and of pre beta 1-HDL produced by hepatic lipase.

The hepatic lipase acting on triglyceride-rich high-density lipoprotein2 (HDL2) induces the formation of pre beta 1-HDL, leaving a residual alpha-migrating HDL particle that was named "remnant-HDL2" (Barrans, A., Collet, X., Barbaras, R., Jaspard, B., Manent, J., Vieu, C., Chap, H., and Perret, B. (1994) J. Biol. Chem. 269, 11572-11577.]. In this study, these two product particles generated by hepatic lipase were isolated by density gradient ultracentrifugation. Particles were first characterized in terms of chemical composition, density, and mass. The pre beta 1-HDL obtained in vitro contain one to two molecules of apoA-I, associated with phospholipids, and free and esterified cholesterol. When compared to triglyceride-rich HDL2, remnant-HDL2 have lost on average one molecule of apoA-I, 60% of triacylglycerols, and 15% of phospholipids. The estimated composition is concordant with the hypothesis of the splitting of a substrate particle into one pre beta 1-HDL and one remnant-HDL2. Spectroscopic studies were carried out to monitor changes in lipid fluidity upon lipolysis. The fluorescence anisotropy was measured using (1,6)-diphenyl-hexa-(1,3, 5)-triene as a probe, and the degree of order was calculated from electron spin resonance spectra using the 5-nitroxy-derivative of stearic acid. Both approaches showed a decreased lipid fluidity in remnant-HDL2, as compared to triglyceride-rich HDL2. The immunoreactivity of apoA-I toward several monoclonal antibodies was assayed as a reflection of changes of apoA-I conformation. In remnant-HDL2, as compared to triglyceride-rich HDL2, a lower reactivity was noted with the 2G11 antibody, which interacts in the NH2 terminal part of apoA-I. Finally, remnant-HDL2 was clearly different from HDL3 with respect to all of the parameters studied, demonstrating that hepatic lipase does not promote the direct conversion of HDL2 to HDL3. Thus, hepatic lipase produces remnant-HDL2 particles, which display modifications of apoA-I conformation and of fluidity of the lipid environment. This newly described HDL2 subfraction may play a major role in the reverse cholesterol transport.

Remnant high density lipoprotein2 particles produced by hepatic lipase display high-affinity binding and increased endocytosis into a human hepatoma cell line (HEPG2).

We had previously shown that hepatic lipase plays a prominent role in promoting the generation of pre-beta HDL particles from triglyceride rich HDL2, leaving an alpha-HDL particle of decreased size that was named "remnant HDL2" [Barrans, A., et al. (1994) J. Biol. Chem. 269, 11572-11577]. Interestingly, this remnant HDL2 was rapidly cleared by the liver, suggesting a particularly high affinity of those remnant HDL2 for liver cells. In the present study, we attempted to characterize the interaction of remnant HDL2 with HepG2 cells, as compared to those of native triglyceride rich HDL2. Two main observations were made. First, while triglyceride rich HDL2 particles were able to bind only the low-affinity binding sites, the remaining particle generated after hepatic lipase lipolysis the remnant HDL2 was further able to bind to the high-affinity binding sites. Competition experiments indicate that these two remnant HDL2 binding sites were the same as the two HDL3 binding sites previously described [Barbaras, R., et al. (1994) Biochemistry 33, 2335-2340]. This is the first observation on the remodeling dependence of HDL binding onto hepatocytes. Second, following binding on those two binding sites, the remnant HDL2 were faster internalized and in higher amounts than the native triglyceride rich HDL2. All together, these observations suggest that the continuous remodeling of HDL induces different binding and internalization characteristics of the HDL particles and that the high-affinity HDL binding sites might trigger the internalization of apo HDL through the low-affinity binding sites.

Structural and functional comparison of HDL from homologous human plasma and follicular fluid. A model for extravascular fluid.

In the preovulatory period, follicular fluid contains only HDL. Biochemical characterization of such lipoproteins showed that follicular fluid HDLs were cholesterol-poor particles compared with serum HDLs, whereas the amount of phospholipids, expressed as percent weight, was significantly higher in follicular fluid HDLs (28.5%) than in serum HDLs (25.0%, P < .05). The amount of apolipoprotein (apo) A-IV per apo A-I was significantly higher in follicular fluid than in serum (0.77 versus 0.58 mg/g apo A-I, P < .02). To explore the role of HDLs as cholesterol acceptors in physiological media, we compared the ability of either whole human follicular fluids or homologous sera to promote cellular cholesterol efflux using Fu5AH rat hepatoma cells. At equivalent concentrations of HDL cholesterol in follicular fluid and in serum, t1/2 values for cholesterol efflux were in the same range. In addition, estimated maximal efflux values were not significantly different in follicular fluid and serum (45.9% and 49.6%, respectively), as were K(m) values (0.064 and 0.071 mmol/L HDL cholesterol respectively). In addition, isolated HDLs displayed the same capacity to promote cellular cholesterol efflux in both media. Thus, the kinetics and dose-response data between these two physiological media showed that HDLs play the major role in cellular cholesterol efflux. The rate of cholesterol esterification, as measured in the presence of cells, was significantly higher in follicular fluid than in serum at constant HDL cholesterol concentrations, whereas the rate of esterified cholesterol transfer toward added LDL was lower. In contrast, in a cell-free system, lecithin:cholesterol acyltransferase activity represented only 26% of that in serum HDL, whereas cholesterol ester transfer protein activities were comparable. In summary, in this particular model, we confirmed the essential role of HDLs as physiological acceptors in the removal of cellular cholesterol.

High-density lipoprotein 3 receptor-dependent endocytosis pathway in a human hepatoma cell line (HepG2).

The internalization of HLD3 into HepG2 cells at 37 degrees C was precisely measured, taking advantage of the previously observed rapid dissociation of HDL3 from its two binding sites [Barbaras, R., et al. (1994) Biochemistry 33, 2335-2340]. We observed a high level of HDL3 internalization (100 ng/mg of cell protein, corresponding to 45.5% of the total HDL3 associated to the cells at 37 degrees C) reaching a plateau at 15 min. Apolipoprotein A-I (the main HDL3 apolipoprotein) associated with dimyristoylphosphatidylcholine (DMPC) complexes was also internalized by HepG2 cells, at levels comparable to those obtained with HDL3 lipid-free apolipoprotein A-I, which can bind only to the HDL3 high-affinity binding site, and displayed a weak internalization (5 ng internalized/mg of cell protein compared to 250 ng/mg for apolipoprotein A-I complexed with DMPC). Clathrin-coated vesicle purification following HDL3 or LDL internalization at 37 degrees C showed radioactivity associated with these vesicles, and further content analysis evidenced the presence of radiolabeled apoA-I and apoB, respectively. Treatment of the cells either by saccharose hypertonic shock or by potassium depletion, in order to block clathrin-coated vesicle formation, completely inhibited HDL3 internalization, as also observed with LDL. Altogether, these observations clearly demonstrate that HDL3 internalization into HepG2 cells occurs through an endocytosis pathway involving an interaction between apolipoprotein A-I and a cell surface protein, leading to the formation of clathrin-coated vesicles.

Identification and quantification of diacylglycerols in HDL and accessibility to lipase.

We have investigated the presence of diacylglycerols in lipoproteins and especially in HDL. Lipoprotein diacylglycerols are very difficult to isolate and to quantify using classical enzymatic techniques, as they are measured in the presence of triacylglycerols and monoacylglycerols. Using a rapid and very sensitive method of gas-liquid chromatography, developed for neutral lipid analysis on an Ultra 1 Hewlett-Packard fused silica capillary column, diacylglycerols (DG) were identified in HDL and classified into five groups: DG 14-16, DG 16-16, DG 16-18, DG 18-18, and DG 18-20. However, their quantitation was difficult due to only partial resolution of molecular species. HDL lipids were submitted to preparative gas-liquid chromatography and diacylglycerols were then silylated using trimethylsilyl reagents. The trimethylsilyl ethers were analyzed by gas-liquid chromatography on a Restek 50 capillary column and were resolved on the basis of carbon number, degree of unsaturation, and double bond positions. The amount of HDL diacylglycerols was twice that of triacylglycerols. The major molecular species of diacylglycerols consisted of 16:0-18:2n-6, 18:0-18:2n-6, and 16:0-18:1n-9 as the major molecular species (33.4, 22.2, and 16.1 mol % of total diacylglycerols, respectively). Using guinea pig cationic pancreatic lipase in order to test the accessibility of diacylglycerols at the surface of HDL, we measured 59% of diacylglycerol hydrolysis, whereas no triacylglycerol hydrolysis was obtained. In addition, most of diacylglycerols having long chain fatty acids, such as 18-20, were completely hydrolyzed, whereas 18-18 and 16-18 were only partially hydrolyzed (64 and 46% respectively). This reflects a different partition of diacylglycerol molecular species between the particle's surface and the lipid core in HDL. This is the first analysis of diacylglycerol molecular species and their distribution in native lipoprotein particles.

Biochemical characterization of pre-beta 1 high-density lipoprotein from human ovarian follicular fluid: evidence for the presence of a lipid core.

In order to isolate pre-beta 1 HDL, we have focused our interest on a particular model, namely, human preovulatory follicular fluid, which contains only HDL as a lipoprotein class as well as a high proportion of pre-beta 1 HDL relative to total HDL (1.5 times more than in homologous plasma) as evidenced by double-dimension gel electrophoresis. Apo A-I in pre-beta 1 HDL represented 17.6% of total apo A-I. Stokes' radii corresponded to 3.42 nm in follicular fluid pre-beta 1 HDL and 3.48 nm in homologous plasma counterparts. After electroelution from agarose, pre-beta 1 HDL were isolated in amounts sufficient to allow characterization by size-exclusion chromatography using HPLC. The estimated apparent molecular mass of these particles is 61.6 kDa. Lipid composition of pre-beta 1 HDL evidenced a low lipid content compared to follicular fluid HDL isolated by ultracentrifugation. Phospholipid composition showed a dramatic decrease in phosphatidylcholines (40.5% of total phospholipids), and the presence of lysophosphatidylcholines and of acidic phospholipids such as phosphatidylserine and phosphatidylinositol (13.6 and 13.7%, respectively). Furthermore, cholesteryl ester and triacylglycerol molecules were quantified by gas-liquid chromatography and represented 8-9% of the pre-beta 1 HDL total weight. Thus, a lipid core is present in pre-beta 1 HDL, which would be compatible with a spherical shape. The follicular fluid appears to be a good model to a better understanding of HDL metabolism.

Hepatic lipase induces the formation of pre-beta 1 high density lipoprotein (HDL) from triacylglycerol-rich HDL2. A study comparing liver perfusion to in vitro incubation with lipases.

High density lipoprotein subfractions with a pre-beta migration play a key role in the reverse cholesterol transport. The origin of these particles is not yet clearly defined. We propose to verify a possible origin of these particles during the catabolism of high density lipoprotein2 (HDL2) by hepatic lipase using two different models. A rat liver perfusion of native human HDL2 in the presence of heparin induced, after 30 min, the formation of the pre-beta 1 HDL subspecies. Human HDL2 enriched with triacylglycerols, perfused in the same conditions, led after 15 min to an enhanced production of this pre-beta 1 HDL population, as compared with the results obtained with native HDL2. A reduction of the alpha-HDL2 fraction was also evident. After perfusion, a similar formation of pre-beta 1 HDL from triacylglycerol-rich HDL2 was observed in absence of heparin. When these HDL2 were incubated in vitro for 120 min at 37 degrees C in the presence of partially purified rat hepatic lipase, the appearance of pre-beta 1 HDL was again found and associated with a decrease in size of the remaining alpha-HDL subfractions as compared with original HDL2. On the contrary, the incubation of the same HDL2 with snake venom phospholipase A2 produced no pre-beta HDL. These results evidence the role of the triacylglycerol lipase activity of hepatic lipase in the formation of pre-beta 1 HDL from triacylglycerol-rich HDL2.

Specific binding of free apolipoprotein A-I to a high-affinity binding site on HepG2 cells: characterization of two high-density lipoprotein sites.

In this paper, we present the first evidence that free apoA-I, without association with lipids, binds only to a high-affinity binding site (Kd = 1.8 microgram/mL, Bmax = 63.12 ng/mL). This is a new binding site of higher affinity (80-100 times) but of lower capacity than the binding sites already described for HDL. This is also the first evidence on HepG2 cells both of a high-affinity site (Kd = 0.685 microgram/mL, Bmax = 39.86 ng/mL) and of a low-affinity site (Kd = 55.65 micrograms/mL, Bmax = 665.45 ng/mL) for HDL. ApoA-I-DMPC complexes also present two binding components comparable to the HDL3 binding sites. This free apoA-I binding is specific, as shown by competition experiments, and allowed us to specifically study this high-affinity site, without interference of the low-affinity one. Kinetic rates of association/dissociation for the high-affinity site were faster than for the low-affinity site (10 and 20 min versus 40 and 30 min, respectively). The kinetic Kd values, derived from association and dissociation rate constants (Kd = 55.14 and 2.91 micrograms/mL), were of similar magnitude as the Kd values calculated by Scatchard analysis. These data confirm that HDL3 binding sites characterized by saturation experiments follow the law of mass action, indicative of ligand-receptor interaction. In summary, HepG2 cells present high HDL3 binding sites which are able to bind free apoA-I in contrast with the low-affinity HDL3 binding sites.

Differential role of apolipoprotein AI-containing particles in cholesterol efflux from adipose cells.

Cholesterol efflux was studied in cultured Ob1771 adipose cells after preloading with LDL cholesterol. Exposure to particles containing apo AII (LpAI) and particles containing apo AI and apo AII (LpAI:AII) isolated from native human plasma, and from HDL2 or HDL3, showed that only LpAI were able to promote cholesterol efflux, despite the fact that both kinds of particles were able to bind to receptor sites within the same range of concentrations (apparent Kd values between 10 and 25 micrograms/ml). During this long-term exposure, LpAI:AII demonstrated a concentration-dependent inhibition (10-60 micrograms/ml) of LpAI-mediated cholesterol efflux from adipose cells under conditions where LpAI:AII did not deliver cholesterol to the cells and where no net change in the distribution of apo AI between LpAI and LpAI:AII was observed. The antagonizing and modulating role of LpAI:AII in preventing cholesterol efflux mediated by LpAI appears not to be related to the lipid composition and cholesterol content of the particles but, rather, appears dependent upon the presence of apo AI in LpAI particles and apo AII in LpAI:AII particles. The actual concentrations of these particles in the interstitial fluid bathing peripheral cells might be critical for the in vivo occurrence of cholesterol efflux.