Selective uptake of cholesteryl esters from various classes of lipoproteins by HepG2 cells.

Selective uptake of cholesteryl esters (CE) from lipoproteins by cells has been extensively studied with high density lipoproteins (HDL). It is only recently that such a mechanism has been attributed to intermediate and low density lipoproteins (IDL and LDL). Here, we compare the association of proteins and CE from very low density lipoproteins (VLDL), IDL, LDL and HDL3 to HepG2 cells. These lipoproteins were either labelled in proteins with 125I or in CE with 3H-cholesteryl oleate. We show that, at any lipoprotein concentration, protein association to the cells is significantly smaller for IDL, LDL, and HDL3 than CE association, but not for VLDL. At a concentration of 20 microg lipoprotein/mL, these associations reveal CE-selective uptake in the order of 2-, 4-, and 11-fold for IDL, LDL, and HDL3, respectively. These studies reveal that LDL and HDL3 are good selective donors of CE to HepG2 cells, while IDL is a poor donor and VLDL is not a donor. A significant inverse correlation (r2 = 0.973) was found between the total lipid/protein ratios of the four classes of lipoproteins and the extent of CE-selective uptake by HepG2 cells. The fate of 3H-CE of the two best CE donors (LDL and HDL3) was followed in HepG2 cells after 3 h of incubation. Cells were shown to hydrolyze approximately 25% of the 3H-CE of both lipoproteins. However, when the cells were treated with 100 microM of chloroquine, a lysosomotropic agent, 85 and 40% of 3H-CE hydrolysis was lost for LDL and HDL3, respectively. The fate of LDL and HDL3-CE in HepG2 cells deficient in LDL-receptor was found to be the same, indicating that the portion of CE hydrolysis sensitive to chloroquine is not significantly linked to LDL-receptor activity. Thus, in HepG2 cells, the magnitude of CE-selective uptake is inversely correlated with the total lipid/protein ratios of the lipoproteins and CE-selective uptake from the two best CE donors (LDL and HDL3) appears to follow different pathways.

Presence of CLA-1 and HDL binding sites on syncytiotrophoblast brush border and basal plasma membranes of human placenta.

It is now known that rapid placental and fetal development is associated with elevated levels of circulating high density lipoprotein (HDL) in pregnant women. The main structure implicated in the maternal-fetal exchange is the syncytiotrophoblast, composed of a brush border membrane (BBM), facing the mother, and a basal plasma membrane (BPM), facing the fetus. In order to understand the mechanisms controlling the placental and fetal supplies of cholesterol, we purified both BBM and BPM and verified the presence of HDL binding sites in these membranes. Binding studies using(125)I-HDL(3)show a single affinity binding site on BPM which has a dissociation constant (K(d)) of 3.45+/-0.43 microg protein/ml and a maximal binding capacity (B(max)) of 5.46+/-1.69 microg protein/mg membrane proteins. In BBM, we observed two affinity binding sites, one with a K(d)of 0.62+/-0.03 microg protein/ml and another one with a K(d)of 6.57+/-0.87 microg protein/ml. Their B(max)values were 0.54+/-0.11 and 2.34+/-0.39 microg of HDL(3)/mg membrane proteins, respectively. CLA-1, a putative HDL-receptor of 85 kDa, was detected on both BPM and BBM, together with two apo A-l binding sites of 110 and 96 kDa on BPM and BBM, respectively. These results provide further evidence that human placenta possesses specific sites for HDL binding, underlining the important role of maternal HDL in the transfer of cholesterol from the maternal circulation to the placenta and the fetus.

Selective uptake of cholesteryl ester from low density lipoprotein is involved in HepG2 cell cholesterol homeostasis.

Low density lipoprotein (LDL) can follow either a holoparticle uptake pathway, initiated by the LDL receptor (LDLr), and be completely degraded, or it can deliver its cholesteryl esters (CE) selectively to HepG2 cells. Although high density lipoprotein-CE selective uptake has been shown to be linked to cell cholesterol homeostasis in nonhepatic cells, there is no available information on the effect of LDL-CE selective uptake on hepatic cell cholesterol homeostasis. In order to define the role of the LDL-CE selective uptake pathway in hepatic cell cholesterol homeostasis, we used a cellular model that expresses constitutively a LDLr antisense mRNA and that shows LDLr activity at 31% the normal level (HepG2-all cells). The addition of a specific antibody anti-LDLr (IgG-C7) reduces LDL protein degradation (LDLr activity) to 7%. This cellular model therefore reflects, above all, LDL-CE selective uptake activity when incubated with LDL. The inactivation of LDLr reduces LDL-protein association by 78% and LDL-CE association by only 43%. The LDL-CE selective uptake was not reduced by the inactivation of LDLr. The activities of the various enzymes involved in cell cholesterol homeostasis were measured in normal and LDLr-deficient cells during incubation in the absence or presence of LDL as a cholesterol source. Essentially, 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl coenzyme A:cholesterol acyltransferase (ACAT) activities responded to LDL in LDLr-deficient cells as well as in normal HepG2 cells. Inhibition of lysosomal hydrolysis with chloroquine abolished the effect measured on ACAT activity in the presence of LDL, suggesting that CE of LDL, but not free cholesterol, maintains cell cholesterol homeostasis. Thus, in HepG2 cells, when LDLr function is virtually abolished, LDL-CE selective uptake is coupled to cell cholesterol homeostasis.

Selective uptake of cholesteryl esters of low-density lipoproteins is mediated by the lipoprotein-binding site in HepG2 cells and is followed by the hydrolysis of cholesteryl esters.

The study described in this paper shows that 125I-labelled low-density lipoproteins (LDL) interact with high- and low-affinity binding sites on human hepatoma (HepG2) cells. The former site is the LDL receptor and the latter is the lipoprotein-binding site (LBS). The association of 125I-labelled LDL and [3H]cholesteryl ethers-LDL with HepG2 cells revealed a 4-fold selective uptake of cholesteryl esters (CE) in a 4 h incubation period, which correlated with the depletion of CE mass in LDL. This selective uptake was not observed when the cells were incubated in the presence of a 100-fold excess of high-density lipoprotein 3, conditions where only the LDL receptor is being monitored. Also, no reduction in uptake was observed in the presence of IgG-C7, an anti-(LDL receptor) monoclonal antibody. Both findings indicate that the selective uptake occurs through the LBS and that the LBS contributes more to the entry of CE from LDL into the cell than does the LDL receptor. The fates of CE entering the cell via the LDL receptor and the LBS were also followed. To achieve this, LDL were labelled with [3H]cholesteryl oleate and the hydrolysis of [3H]cholesteryl oleate was monitored. The results indicated that 45% of the CE were hydrolysed after a 4 h incubation period, irrespective of the site of entry. Chloroquine (100 microM) was shown to inhibit hydrolysis, indicating that lysosomal enzymes were responsible for the hydrolysis of LDL-CE, whichever pathway was used. Thus our results reveal, for the first time, that the mass of CE entering the cell via the LBS is substantial and that hydrolysis of CE is by lysosomal enzyme activity. Overall, this suggests that the LBS has significant physiological importance.

Comparative ultrastructure of submaxillary salivary glands from mice treated with cytosine arabinoside, cyclocytidine, and anhydro-ara-5-fluorocytidine.

The effects of cytosine arabinoside (ara-C), cyclocytidine (cyclo-C), and anhydro-ara-5-fluorocytidine (AAFC) on the ultrastructure of the serous and mucous cells of the submaxillary salivary glands were evaluated in normal BDF1 mice. Rapid loss of zymogen granules from serous cells and a discharge of mucous cell contents were observed within the first hour after administration of a single iv dose of cyclo-C. Serous cells were essentially devoid of granules (1-3 mum) within 3 hours; regranulation observed initially at 6 hours produced a population of microgranules (approximately 0.8 mum) by 24 hours; most serous cells contained a reduced complement of small granules through 96 hours after cyclo-C. Mucous cells recovered secretory content within 3-6 hours after drug administration. Neither ara-C nor AAFC produced zymogen-granule or mucous discharge or other cytopathologic effects in the murine submaxillary salivary glands. Thus, it can be postulated that cyclo-C itself is the secretogogue. The ultrastructural changes observed in this study provide a cytopathologic correlate of the salivary gland dysfunctions reported to follow the administration of cyclo-C to man.