Relación entre Helicobacter pylori y patologías digestivas altas por biopsia y endoscopía en un hospital general en Ica, Perú / Relationship between Helicobacter pylori and high digestive pathologies for biopsy and endoscopy in general hospital of Ica, Peru

Objetivo: Determinar la relación entre Helicobacter pylori y patologías digestivas altas mediante biopsia dirigida por endoscopia en el Hospital Regional de Ica, diciembre 2016 y enero 2017. Material y métodos: Se realizó un estudio observacional descriptivo transversal, cuya muestra estuvo conformada por 228 pacientes escogidos de forma aleatoria, que acudieron a consultorio de procedimientos endoscópicos gastrointestinales del Hospital Regional de Ica durante diciembre del 2016 y enero del 2017. Para determinar la relación entre Helicobacter pylori y patologías digestivas altas, creamos un instrumento de recolección de datos, se realizó el análisis exploratorio, univariado y bivariado (OR, Chi² y ANOVA), en la base de datos SPSS 22. Resultados: La relación entre presencia de Helicobacter pylori y Gastritis crónica (p=0.00) fue significativa, no se encontró relación con otra patología. El proceder de lugares diferentes de Ica se encontró asociado a infección por Helicobacter pylori, siendo un factor protector (OR: 0.25, p=0.04), el ingerir verduras y frutas tienen asociación significativa con la infección, comportándose como factores protectores (p<0.05), y el consumo de carnes y tubérculos incrementan la probabilidad de infectarse con el microorganismo (ORc: 2.63 y ORt: 2.29, p<0.05). Conclusiones: Existe relación entre Gastritis crónica e infección por Helicobacter pylori, consumir verduras y frutas disminuye la probabilidad de infección, mientras que consumir abundantes carnes y tubérculos incrementan el riesgo de infección; y residir fuera de Ica es un factor que disminuye la probabilidad de infectarse. (AU)

Objetive: To determine the relationship between the presence of Helicobacter pylori and upper digestive pathologies by endoscopy-guided biopsy at the Regional Hospital of Ica, December 2016 and January 2017. Materials and Methods: A cross-sectional, observational, descriptive study was performed. 228 randomly selected patients who visited the gastrointestinal endoscopic procedures of the Regional Hospital of Ica during the months of December 2016 and January 2017. To determine the relationship between the presence of Helicobacter pylori and upper digestive pathologies, we created an instrument (OR, Chi² and ANOVA) were analyzed in the SPSS 22 database. Results: The relationship between the presence of Helicobacter pylori and chronic gastritis (p = 0.00) is significant, No relationship was found with other pathology. The procedure of different sites of Ica is associated with Helicobacter infection, being a protective factor (OR: 0.25, p = 0.04), ingesting vegetables and fruits have a significant association with infection, behaving as protective factors (p <0.05), And the consumption of meats and tubers increases the probability of being infected with the microorganism (ORc: 2.63 and ORt: 2.29, p <0.05).Conclusions: There is a relationship between chronic Gastritis and Helicobacter pylori infection, consuming vegetables and fruits reduces the probability of infection, while consuming abundant meats and tubers increase the risk of infection; And residing outside of Ica is a factor that decreases the probability of becoming infected. (AU)

Characterization of oxidized low-density lipoprotein-induced hormesis-like effects in osteoblastic cells.

Epidemiological studies indicate that patients suffering from atherosclerosis are predisposed to develop osteoporosis. Atherogenic determinants such as oxidized low-density lipoprotein (oxLDL) particles have been shown both to stimulate the proliferation and promote apoptosis of bone-forming osteoblasts. Given such opposite responses, we characterized the oxLDL-induced hormesis-like effects in osteoblasts. Biphasic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reductive activity responses were induced by oxLDL where low concentrations (10-50 microg/ml) increased and high concentrations (from 150 microg/ml) reduced the MTT activity. Cell proliferation stimulation by oxLDL partially accounted for the increased MTT activity. No alteration of mitochondria mass was noticed, whereas low concentrations of oxLDL induced mitochondria hyperpolarization and increased the cellular levels of reactive oxygen species (ROS). The oxLDL-induced MTT activity was not related to intracellular ROS levels. OxLDL increased NAD(P)H-associated cellular fluorescence and flavoenzyme inhibitor diphenyleneiodonium reduced basal and oxLDL-induced MTT activity, suggesting an enhancement of NAD(P)H-dependent cellular reduction potential. Low concentrations of oxLDL reduced cellular thiol content and increased metallothionein expression, suggesting the induction of compensatory mechanisms for the maintenance of cell redox state. These concentrations of oxLDL reduced osteoblast alkaline phosphatase activity and cell migration. Our results indicate that oxLDL particles cause hormesis-like response with the stimulation of both proliferation and cellular NAD(P)H-dependent reduction potential by low concentrations, whereas high concentrations lead to reduction of MTT activity associated with the cell death. Given the effects of low concentrations of oxLDL on osteoblast functions, oxLDL may contribute to the impairment of bone remodeling equilibrium.

[Health professionals in part-time employment: the challenge of balancing work and family]. / Professionnelles de la santé en précarité d'emploi: le défi de la conciliation travail-famille.

Looking at the case of occasional part-time nurses, this study highlights the difficulties in balancing work and family that are inherent in nonstandard jobs. Eight focus groups were held, involving 48 nurses in 4 regions of Quebec. Analysis of the data collected reveals that nurses "on call" are particularly affected by overwork and experience great difficulty in balancing their work and family obligations. The participants proposed a variety of solutions, such as establishing day care centres adapted to the needs of nurses on call and instituting a timetable grid for occasional part-time nurses so that they can plan their work hours.

Low and high density lipoprotein metabolism in primary cultures of hepatic cells from normal and apolipoprotein E knockout mice.

Apolipoprotein E (apoE) plays a major role in lipoprotein metabolism by mediating the binding of apoE-containing lipoproteins to receptors. The role of hepatic apoE in the catabolism of apoE-free lipoproteins such as low density lipoprotein (LDL) and high density lipoprotein-3 (HDL(3)) is however, unclear. We analyzed the importance of hepatic apoE by comparing human LDL and HDL(3) metabolism in primary cultures of hepatic cells from control C57BL/6J and apoE knockout (KO) mice. Binding analysis showed that the maximal binding capacity (Bmax) of LDL, but not of HDL(3), is increased by twofold in the absence of apoE synthesis/secretion. Compared to control hepatic cells, LDL and HDL(3) holoparticle uptake by apoE KO hepatic cells, as monitored by protein degradation, is reduced by 54 and 77%, respectively. Cleavage of heparan sulfate proteoglycans (HSPG) by treatment with heparinase I reduces LDL association by 21% in control hepatic cells. Thus, HSPG alone or a hepatic apoE-HSPG complex is partially involved in LDL association with mouse hepatic cells. In apoE KO, but not in normal hepatic cells, the same treatment increases LDL uptake/degradation by 2.4-fold suggesting that in normal hepatic cells, hepatic apoE increases LDL degradation by masking apoB-100 binding sites on proteoglycans. Cholesteryl ester (CE) association and CE selective uptake (CE/protein association ratio) from LDL and HDL(3) by mouse hepatic cells were not affected by the absence of apoE expression. We also show that 69 and 72% of LDL-CE hydrolysis in control and apoE KO hepatic cells, respectively, is sensitive to chloroquine revealing the importance of a pathway linked to lysosomes. In contrast, HDL(3)-CE hydrolysis is only mediated by a nonlysosomal pathway in both control and apoE KO hepatic cells. Overall, our results indicate that hepatic apoE increases the holoparticle uptake pathway of LDL and HDL(3) by mouse hepatic cells, that HSPG devoid of apoE favors LDL binding/association but impairs LDL uptake/degradation and that apoE plays no significant role in CE selective uptake from either human LDL or HDL(3) lipoproteins.

Structure-function relationships of human apolipoprotein D an immunochemical analysis.

Apolipoprotein D (apoD), a 169 amino acid member of the lipocalin family, is thought to be a transporter of small, hydrophobic ligands. A panel of 10 anti-apoD monoclonal antibodies (mAbs) was prepared and characterized in order to define apoD structure-function relationships. An apoD epitope map was constructed based on reactivity of the mAbs with apoD fragments. Three mAbs react with epitopes between apoD residues 7-78, seven mAbs with epitopes between residues 128-169, one mAb recognizes an epitope that straddles residues 99-102 and one mAb is specific for an epitope composed of non-contiguous apoD residues. Several pairs of mAbs whose respective epitopes are widely separated in apoD primary structure can compete for binding to immobilized apoD. This would be consistent with the compact beta-barrel tertiary structure that apoD is thought to adopt. None of the mAbs block the interaction of apoD with pregnenolone, a putative physiological ligand for apoD.

Low- and high-density lipoprotein metabolism in HepG2 cells expressing various levels of apolipoprotein E.

To determine the importance of hepatic apolipoprotein (apo) E in lipoprotein metabolism, HepG2 cells were transfected with a constitutive expression vector (pRc/CMV) containing either the complete or the first 474 base pairs of the human apoE cDNA inserted in an antisense orientation, for apoE gene inactivation, or the full-length human apoE cDNA inserted in a sense orientation for overexpression of apoE. Stable transformants were obtained that expressed 15, 24, 226, and 287% the apoE level of control HepG2 cells. The metabolism of low-density lipoprotein (LDL) and high-density lipoprotein-3 (HDL(3)), two lipoprotein classes following both holoparticle and cholesteryl esters (CE)-selective uptake pathways, was compared between all these cells. LDL-protein degradation, an indicator of the holoparticle uptake, was greater in low apoE expressing cells than in control or high expressing cells, while HDL(3)-protein degradation paralleled the apoE levels of the cells (r(2) = 0.989). LDL- and HDL(3)-protein association was higher in low apoE expressing cells compared to control cells. In opposition, LDL- and HDL(3)-CE association was not different from control cells in low apoE expressing cells but rose in high apoE expressing cells. In consequence, the CE-selective uptake (CE/protein association ratio) was positively correlated with the level of apoE expression in all cells for both LDL (r(2) = 0.977) and HDL(3) (r(2) = 0.998). We also show that, although in normal and low apoE expressor cells, 92% of LDL- and 80% HDL(3)-CE hydrolysis is sensitive to chloroquine suggesting a pathway linked to lysosomes for both lipoproteins, cells overexpressing apoE lost 60% of chloroquine-sensitive HDL(3)-CE hydrolysis without affecting that of LDL-CE. Thus, the level of apoE expression in HepG2 cells determines the fate of LDL and HDL(3).

Implication of ATP and sodium in arachidonic acid incorporation by placental syncytiotrophoblast brush border and basal plasma membranes in the human.

The human placental syncytiotrophoblast is the main site of exchange of nutrients and minerals between the mother and her fetus. In order to characterize the placental transport of some fatty acids, we studied the incorporation of arachidonic acid, a fetal primordial fatty acid, in purified bipolar syncytiotrophoblast brush border (BBM) and basal plasma membranes (BPM) from human placenta. The basal arachidonic acid incorporation in BBM and BPM was time dependent and reached maximal values of 0.75+/-0.10 and 0.48+/-0.18 pmol/mg protein, respectively, after 2.5 min. The presence of adenosine triphosphate (ATP) (3 m m) increases significantly the maximal incorporation of arachidonic acid by sixfold (4.75+/-0.35 pmol/mg) and ninefold (4.40+/-0.84 pmol/mg) in BBM and BPM, respectively. Moreover, an increase in the arachidonic acid incorporation was also obtained in the presence of sodium where the values achieved 7.68+/-0.98 (10x) and 6.53 pmol/mg (13.6x) for BBM and BPM, respectively. We also showed that the combination of both Na(+)and ATP increases significantly the maximal incorporation of arachidonic acid in BPM to 7.89+/-0.15 pmol/mg protein, while in BBM it did not modify its incorporation (8.18+/-0.25 pmol/mg protein), as compared to the presence of sodium alone. Our results demonstrate that arachidonic acid is incorporated by both placental syncytiotrophoblast membranes, and is ATP and sodium-linked. However, different mechanisms seem to be involved in this fatty acid incorporation through BBM and BPM, since the presence of Na(+)or ATP increased it, while the association of these two elements increased it only in BPM. We also demonstrated by osmolarity experiments that both membranes bind arachidonic acid, potentially involving one or more fatty acids binding proteins.

Analysis of low-density lipoprotein catabolism by primary cultures of hepatic cells from normal and low-density lipoprotein receptor knockout mice.

Low-density lipoproteins (LDL) are taken up by LDL receptor (LDLr)-dependent and -independent pathways; the role and importance of the latest being less well defined. We analyzed the importance of these pathways in the mouse by comparing LDL binding to primary cultures of hepatocytes from LDLr knockout (LDLr KO) and normal C57BL/6J mice. Saturation curve analysis shows that (125)I-LDL bind specifically to normal and LDLr KO mouse hepatocytes with similar dissociation constants (K(d)) (31.2 and 22.9 microg LDL-protein/ml, respectively). The maximal binding capacity (B(max)) is, however, reduced by 48% in LDLr KO mouse hepatocytes in comparison to normal hepatocytes. Conducting the assay in the presence of a 200-fold excess of high-density lipoprotein-3 (HDL3) reduced by 39% the binding of (125)I-LDL to normal hepatocytes and abolished the binding to the LDLr KO mouse hepatocytes. These data indicate that in normal mouse hepatocytes, the LDLr is responsible for approximately half of the LDL binding while a lipoprotein binding site (LBS), interacting with both LDL and HDL3, is responsible for the other half. It can also be deduced that both receptors/sites have a similar affinity for LDL. The metabolism of LDL-protein and cholesteryl esters (CE) was analyzed in both types of cells. (125)I-LDL-protein degradation was reduced by 95% in LDLr KO hepatocytes compared to normal hepatocytes. Comparing the association of (125)I-LDL and (3)H-CE-LDL revealed a CE-selective uptake of 35.6- and 22-fold for normal and LDLr KO mouse hepatocytes, respectively. Adding a 200-fold excess of HDL3 in the assay reduced by 71% the CE-selective uptake in LDLr KO hepatocytes and by 96% in normal hepatocytes. This indicates that mouse hepatocytes are able to selectively take up CE from LDL by the LBS. The comparison of LDL-CE association also showed that the LBS pathway provides 5-fold more LDL-CE to the cell than the LDLr. Overall, our results indicate that in mouse hepatocytes, LDLr is almost completely responsible for LDL-protein degradation while the LBS is responsible for the major part of LDL-CE entry by a CE-selective uptake pathway.

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.

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.

Low density lipoprotein-receptor plays a major role in the binding of very low density lipoproteins and their remnants on HepG2 cells.

The binding to HepG2 cells of very low density lipoproteins (VLDL) and their remnants (IDL) was alternatively, in the past, attributed to the low density lipoprotein receptor (LDLr) or to an apoE-specific receptor. In order to resolve this issue, we have compared the binding of those lipoproteins labelled with iodine-125 to normal and LDLr deficient HepG2 cells. Those deficient cells were obtained by a constitutive antisense strategy and their LDLr level is 14% the level of normal HepG2 cells. By saturation curve analysis, we show that VLDL and IDL bind to high and low affinity sites on cells. The low affinity binding was eliminated by conducting the assay in presence of a 200-fold excess of HDL3 respective to the concentrations of 125I-labelled VLDL and IDL. For 125I-VLDL high affinity binding to normal HepG2 cells, we found a dissociation constant (Kd) of 21.2 +/- 3.7 micrograms prot./ml (S.E., N = 5) and a maximal binding capacity (Bmax) of 0.0312 +/- 0.0063 microgram prot./mg cell prot, while we have measured a Kd of 5.3 +/- 0.8 and a Bmax of 0.0081 +/- 0.0014 with LDLr deficient cells. This indicates that LDLr is responsible for 74% of VLDL binding to HepG2 cells and that the non-LDLr high affinity receptor has a higher affinity for VLDL than LDLr. A 53% loss of 125I-IDL binding capacity was measured with LDLr deficient cells compared with normal cells (Bmax: 0.028 +/- 0.005 versus 0.059 +/- 0.006), while no significant statistical difference was found between affinities. The study shows that the LDLr is almost the only contributor in VLDL binding, while it shares IDL binding capacity with another high affinity receptor. The physiological importance of LDLr is confirmed by an almost equivalent loss of IDL and VLDL degradation in LDLr deficient cells.

Impact of a cholesterol enriched diet on maternal and fetal plasma lipids and fetal deposition in pregnant rabbits.

Pregnancy is associated with a hypercholesterolemic and a hyperlipidemic state. The totality of the essential fatty acids and 50% of the lipids needed by the fetus are transferred by the placenta from the maternal circulation. The hypothesis of this study is that an augmentation of the maternal plasmatic cholesterol is modifying the fetal lipids accumulation and development during rabbit pregnancy. To demonstrate the impact of a cholesterol enriched diet on plasma lipids during rabbit's pregnancy and on their fetus, we have established two groups: control and hypercholesterolemic rabbits (fed with a 0.2% cholesterol diet). Blood samples were collected before mating and at each trimester of pregnancy for analysis of lipid fractions and their lipoproteins. Plasma analysis shows that starting the 10th day of pregnancy the concentration of total-cholesterol and lipoproteins decreases for both groups. We have demonstrated that for the hypercholesterolemic group, concentrations of total-cholesterol (631%) and lipoproteins are significantly higher at the end of pregnancy than those for the control group. For both groups, after 20 days of pregnancy, triglycerides metabolism was biphasic showing a significant increase followed by a diminution in their concentration. In both groups, free fatty acids increases significantly at the end of the pregnancy (537.5% for the control group and 462.5% for the hypercholesterolemic group). Furthermore, the offsprings of hypercholesterolemic dams manifest a lower birth weight (15.5%) than those of control group. Our results demonstrate that a cholesterol enriched diet modifies greatly the fetal development and lipid metabolism during rabbit's pregnancy. These modifications could be useful for the understanding of the interaction between diet and fetal development in rabbit and probably during human pregnancy.

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.

Uptake and fate of class B scavenger receptor ligands in HepG2 cells.

Class B scavenger receptors (SR-Bs) interact with native, acetylated and oxidized low-density lipoprotein (LDL, AcLDL and OxLDL), high-density lipoprotein (HDL3) and maleylated BSA (M-BSA). The aim of this study was to analyze the catabolism of CD36- and LIMPII-analogous-1 (CLA-1), the human orthologue for the scavenger receptor class B type I (SR-BI), and CD36 ligands in HepG2 (human hepatoma) cells. Saturation binding experiments revealed moderate-affinity binding sites for all the SR-B ligands tested with dissociation constants ranging from 20 to 30 microg.mL-1. Competition binding studies at 4 degrees C showed that HDL and modified and native LDL share common binding site(s), as OxLDL competed for the binding of 125I-LDL and 125I-HDL3 and vice versa, and that only M-BSA and LDL may have distinct binding sites. Degradation/association ratios for SR-B ligands show that LDL is very efficiently degraded, while M-BSA and HDL3 are poorly degraded. The modified LDL degradation/association ratio is equivalent to 60% of the LDL degradation ratio, but is three times higher than that of HDL3. All lipoproteins were good cholesteryl ester (CE) donors to HepG2 cells, as a 3.6-4.7-fold CE-selective uptake ([3H]CE association/125I-protein association) was measured. M-BSA efficiently competed for the CE-selective uptake of LDL-, OxLDL-, AcLDL- and HDL3-CE. All other lipoproteins tested were also good competitors with some minor variations. Hydrolysis of [3H]CE-lipoproteins in the presence of chloroquine demonstrated that modified and native LDL-CE were mainly hydrolyzed in lysosomes, whereas HDL3-CE was hydrolyzed in both lysosomal and extralysosomal compartments. Inhibition of the selective uptake of CE from HDL and native modified LDL by SR-B ligands clearly suggests that CLA-1 and/or CD36 are involved at least partially in this process in HepG2 cells.

Effect of reduced low-density lipoprotein receptor level on HepG2 cell cholesterol metabolism.

Low-density lipoproteins (LDL) are taken up by both LDL receptor (LDLr)-dependent and -independent pathways. In order to determine the importance of these pathways in the activity of the various enzymes that are important in maintaining the cellular cholesterol level in hepatic cells, we created HepG2 cells expressing lower levels of LDLr. Thus HepG2 cells were transfected with a constitutive expression vector (pRc/CMV) containing a fragment of LDLr cDNA inserted in an antisense manner. Stable transformants were obtained that showed significant reductions of 42, 72 and 85% of LDLr protein levels compared with the control, as demonstrated by immunoblotting and confirmed by the LDL binding assay. The best inactivation was achieved with the construct containing the first 0.7 kb of LDLr cDNA. Incubating the different HepG2 cell subtypes with LDL showed similar association of apolipoprotein B (apo B) or cholesteryl esters from LDL with the cells, indicating that the LDLr deficiency did not significantly affect LDL uptake by the cell. However, apoB degradation was reduced significantly by 71-82% in the most LDLr-deficient HepG2 cells. We also found that 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoA red) activity is significantly increased by 32-35% in HepG2 cells expressing very low levels of LDLr that also demonstrate a significant decrease of 20% in acyl-CoA:cholesterol acyltransferase (ACAT) activity. However, these effects are moderate compared with those observed when cells were incubated in lipoprotein-depleted medium, where a >900% increase in HMGCoA red activity and a loss of 60% of ACAT activity was observed. Thus, in HepG2 cells, different levels of LDLr affect LDL-apoB degradation, but have very little effect on LDL association, HMGCoA red and ACAT activities, revealing that LDLr is more important in the clearance of LDL-apoB than in HepG2 cell cholesterol homoeostasis, a role that should be attributable to both LDLr-dependent and -independent pathways.

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.

The selective uptake of the cholesteryl esters of low density lipoproteins parallels the activity of protein kinase C.

The analysis of the association of (125)I-LDL and [(3)H]cholesteryl ethers (CEt)-LDL with HepG2 cells revealed a selective uptake of cholesteryl esters (CE) of the LDL, as in the order of three-fold more CE were associated with the cells than LDL-proteins for an incubation of 4 h. To determine if a trans-signalling pathway is involved in this selective uptake, HepG2 cells were pre-treated for 2 h with either a Protein Kinase A activator [8-(4-chlorophenylthioadenosine 3'-5' cyclic monophosphate (CPT-cAMP)] or a Protein Kinase C activator [phorbol 12-myristate 13-acetate (PMA)]. We found that CPT-cAMP had a minimal effect, while PMA was able to significantly increase the selective uptake of the CE of LDL. Indeed, upon a 2 h pre-incubation of HepG2 cells with PMA at a concentration of 160 microM, an increase of more than 3-fold in CE selective uptake was registered and was shown to occur by the lipoprotein binding sites (LBS) of HepG2 cells. Also, an incubation of the cells with 100 nM calphostin C, an inhibitor of protein kinase C, decreased the selective uptake by 41%. The effect of PMA is not abolished by either cycloheximide or actinomycin D. However, cycloheximide was shown to potentiate the effect of PMA on the LBS activity, suggesting that a protein which synthesis is affected by cycloheximide is involved in maintaining the LBS activity low. Our results show that the HepG2 cell activity of CE selective uptake parallels the activity of Protein Kinase C and suggest that the LBS could be a G-protein linked receptor.

Analysis of the selective uptake of the cholesteryl ester of human intermediate density lipoproteins by HepG2 cells.

We have recently shown by the analysis of the association to HepG2 cells of human intermediate density lipoproteins (IDL) either labeled in cholesteryl ester (CE) with [3H]cholesteryl ethers (CEt) or in proteins by iodine-125 that the CE of IDL are selectively taken up by these cells. Our results also revealed that the addition of a sufficient quantity of HDL3 abolishes the binding of IDL to the 'Lipoprotein binding site' (LBS) and also the CE-selective uptake. This suggested that the LBS mediates this uptake (Brissette and Falstrault (1992) Biochim. Biophys. Acta. 1165, 84-92). This study was undertaken to analyze further the mechanism of the selective uptake of the CE of IDL by HepG2 cells to determine if the LBS is directly or indirectly involved. We show that the different labeling had no effect on the binding affinity of IDL to HepG2 cells. To verify that the apolipoprotein moiety of HDL3 was responsible for the abolishment of the CE selective uptake, we have studied the effect of free apoA-I and apoA-II on the association of IDL. Our results demonstrate that apoA-I and apoA-II are approximately 10-times better than HDL3 or apoA-I liposomes in abolishing the selective uptake of the CE from IDL. We also show that this correlates with a more efficient reduction of the binding of 125I-IDL to HepG2 cells by free apoA-I compared to apoA-I associated with lipids. Thus free apoA-I interfere with the binding of IDL to the LBS and free apolipoproteins have a better capacity to saturate the LBS than lipoproteins. Also, we found no evidence for the transfer of CE from the labeled IDL to HDL3 or to apolipoproteins used to abolish the interaction of IDL to the LBS. Thus our results indicate that the LBS is directly responsible for the selective uptake of CE.

Analysis of the lipoprotein binding site of rat liver membranes.

Intermediate density lipoproteins (IDL) were shown to bind to high- and low-affinity binding sites on rat liver membranes. The low-affinity sites were named lipoprotein binding sites (LBS), since they bind all classes of lipoproteins. This study was undertaken to further characterize the interaction of 125I-labelled IDL with the LBS of rat liver membranes to determine the chemical nature of the LBS. We found that the binding of IDL to the LBS is insensitive to EDTA and sensitive to heparin and that it is present on plasma membranes. Also, membranes were pretreated with various enzymes that have an effect on the membrane constituents, and the activity of the LBS on these treated membranes was determined. Our results reveal that the LBS of rat liver membranes is insensitive to heparinase I, chondroitinase ABC, and phospholipase C, while it is partially sensitive to phospholipase A2 and sensitive to proteases and heat. Rat liver membrane proteins were solubilized with Triton X-100, reconstituted in liposomes, and analyzed for their ability to bind lipoproteins. 125I-labelled IDL were shown to bind to high- and low-affinity sites that are similar, in affinity and specificity, to the ones observed with intact rat liver membranes, indicating that a LBS activity is detectable on these liposomes. We found that the binding capacity of low-affinity sites in liposomes containing either no protein or containing proteins solubilized from Escherichia coli membranes is five times weaker than low-affinity sites in liposomes containing liver membrane proteins. Thus, a protein solubilized from rat liver membranes has LBS activity when reconstituted in liposomes. Taken altogether our results provide new information on the binding of IDL to the LBS and indicate that the LBS activity is in part mediated by a protein. Thus, the LBS appears to be a bona fide receptor.