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

1998 ◽  
Vol 329 (1) ◽  
pp. 81-89 ◽  
Author(s):  
Lahoucine IZEM ◽  
Eric RASSART ◽  
Lassana KAMATE ◽  
Louise FALSTRAULT ◽  
David RHAINDS ◽  
...  
Keyword(s):  
Hepg2 Cell ◽  
Hepg2 Cells ◽  
Cholesterol Metabolism ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Constitutive Expression ◽  
Density Lipoprotein ◽  
Low Density ◽  
Apo B ◽  
Acat Activity

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.

scholarly journals Ligand selectivity of 105 kDa and 130 kDa lipoprotein-binding proteins in vascular-smooth-muscle-cell membranes is unique

1996 ◽  
Vol 317 (1) ◽  
pp. 297-304 ◽  
Author(s):  
Valery N. BOCHKOV ◽  
Vsevolod A. TKACHUK ◽  
Maria P. PHILIPPOVA ◽  
Dimitri V. STAMBOLSKY ◽  
Fritz R. BÜHLER ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Binding Proteins ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Dextran Sulphate ◽  
Apo B ◽  
Ligand Selectivity ◽  
Lipoprotein Binding

Using ligand blotting techniques, with low-density lipoprotein (LDL) as ligand, we have previously described the existence of atypical lipoprotein-binding proteins (105 kDa and 130 kDa) in membranes from human aortic medial tissue. The present study demonstrates that these proteins are also present in membranes from cultured human (aortic and mesenteric) and rat (aortic) vascular smooth-muscle cells (VSMCs). To assess the relationship of 105 and 130 kDa lipoprotein-binding proteins to known lipoprotein receptors, ligand binding specificity was studied. We tested effects of substances known to antagonize ligand binding to either the LDL [apolipoprotein B,E (apo B,E)] receptor (dextran sulphate, heparin, pentosan polysulphate, protamine, spermine, histone), the scavenger receptor (dextran sulphate, fucoidin), the very-low-density-lipoprotein (VLDL) receptor [receptor-associated protein (RAP)], or LDL receptor-related protein (RAP, α2-macroglobulin, lipoprotein lipase, exotoxin-A). None of these substances, with the exception of dextran sulphate, influenced binding of LDL to either 105 or 130 kDa proteins. Sodium oleate or oleic acid, known stimuli for the lipoprotein binding activity of the lipolysis-stimulated receptor, were also without effect. LDL binding to 105 and 130 kDa proteins was inhibited by anti-LDL (apo B) antibodies. LDL and VLDL bound to 105 and 130 kDa proteins with similar affinities (蝶50 μg/ml). The unique ligand selectivity of 105 and 130 kDa proteins supports the existence of a novel lipoprotein-binding protein that is distinct from all other currently identified LDL receptor family members. The similar ligand selectivity of 105 and 130 kDa proteins suggests that they may represent variant forms of an atypical lipoprotein-binding protein.

Effect of taurine on cholesterol metabolism in hamsters: Up-regulation of low density lipoprotein (LDL) receptor by taurine

Life Sciences ◽  
2002 ◽  
Vol 70 (20) ◽  
pp. 2355-2366 ◽  
Author(s):  
Shigeru Murakami ◽  
Yukiko Kondo ◽  
Yoshihisa Toda ◽  
Hideaki Kitajima ◽  
Kazuya Kameo ◽  
...  
Keyword(s):  
Cholesterol Metabolism ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density

Short-term effects of ACTH on the low-density lipoprotein receptor mRNA level in rat and hamster adrenals

1991 ◽  
Vol 6 (3) ◽  
pp. 223-230 ◽  
Author(s):  
J.-G. Lehoux ◽  
A. Lefebvre
Keyword(s):  
Cholesterol Metabolism ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Cholesterol Content ◽  
Low Density ◽  
Short Term ◽  
Hmg Coa Reductase ◽  
Receptor Mrna ◽  
Coa Reductase

ABSTRACT Low-density lipoprotein (LDL) receptor mRNA was found in both rat and hamster adrenals. Within 30 min after ACTH administration a significant increase in the levels of both LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) mRNAs was observed in rat adrenals; these levels remained increased for up to 240 min. The increase in the levels of LDL receptor and HMG-CoA reductase mRNAs produced by ACTH was reduced by co-administration of aminoglutethimide while, at the same time, the adrenal cholesterol content of rats treated with both aminoglutethimide and ACTH was significantly increased compared with that in groups treated with ACTH alone. Cycloheximide also induced increased levels of rat adrenal mRNAs for LDL receptor and HMG-CoA reductase, but this effect was not additive with that of ACTH. These results suggest that, in the rat, the short-term effect of ACTH on the levels of mRNAs for the LDL receptor and HMG-CoA reductase is similarly controlled and might be mediated through changes in the adrenal cholesterol content. In the hamster adrenal, however, no significant fluctuations were found in the level of LDL receptor mRNA, although a marked increase was found in the level of HMG-CoA reductase mRNA, 2 h after ACTH administration. This indicates that an important effect of ACTH on cholesterol metabolism in the hamster adrenal is at the level of HMG-CoA reductase. In the hamster, therefore, where the main source of cholesterol for the adrenal gland is de-novo synthesis, it seems that a complex mechanism is involved in the control of LDL receptor gene expression.

scholarly journals The human asialoglycoprotein receptor is a possible binding site for low-density lipoproteins and chylomicron remnants

1991 ◽  
Vol 276 (1) ◽  
pp. 79-87 ◽  
Author(s):  
E Windler ◽  
J Greeve ◽  
B Levkau ◽  
V Kolb-Bachofen ◽  
W Daerr ◽  
...  
Keyword(s):  
Cell Density ◽  
Hepg2 Cells ◽  
High Cell Density ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Asialoglycoprotein Receptor ◽  
Low Density ◽  
High Cell ◽  
Chylomicron Remnants

Binding and internalization of chylomicron remnants from rat mesenteric lymph by HepG2 cells was inhibited by both excess remnants and low-density lipoprotein (LDL) to the same extent. Ligand blots revealed binding of remnants and LDL to the LDL receptor. Measures regulating LDL receptor activity greatly influenced the binding of remnants: ethinyloestradiol, the hydroxymethylglutaryl-CoA reductase inhibitor pravastatin and the absence of LDL all increased binding, whereas high cell density or the presence of LDL decreased binding. Also, asialofetuin, asialomucin, the neoglycoprotein galactosyl-albumin and an antibody against the asialoglycoprotein receptor all decreased substantially the binding of remnants. At high cell density, binding internalization and degradation of chylomicron remnants was inhibited by up to 70-80%, yet binding of LDL was inhibited by no more than 20-30%. In cross-competition studies, the binding of 125I-asialofetuin was efficiently competed for by asialofetuin itself or by the antibody, and also by LDL and remnants, yet remnants displayed an approx. 100-fold higher affinity than LDL. Likewise, remnants of human triacylglycerol-rich lipoproteins and asialofetuin interfered with each others' binding to HepG2 cells or human liver membranes. It is concluded that the LDL receptor mediates the internalization of chylomicron remnants into hepatocytes depending on its activity, according to demand for cholesterol. Additionally, the asialoglycoprotein receptor may contribute to the endocytosis of LDL, but predominantly of chylomicron remnants.

Mutations that affect membrane receptor for LDL are useful for studying normal receptor function

1982 ◽  
Vol 243 (1) ◽  
pp. E5-E14
Author(s):  
R. G. Anderson
Keyword(s):  
Cell Biology ◽  
Cholesterol Metabolism ◽  
Plasma Cholesterol ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Cholesterol Transport ◽  
Low Density ◽  
Receptor Function ◽  
Coated Pits

Low-density lipoprotein (LDL), the major plasma cholesterol transport protein, is taken up by cells through a receptor-mediated process. After internalization through specialized segments of the cell surface called coated pits, the LDL is degraded in the lysosome and the released cholesterol is used by cells to meet various metabolic needs. The discovery of the LDL receptor and the studies of its function have provided new insights into both the biochemical aspects of cholesterol metabolism and the cell biology of receptor-mediated endocytosis. Of paramount importance in all of these studies has been the availability of human cells that express one or more allelic mutations that affect the function of the LDL receptor. These mutations have been valuable for assessing normal receptor function. Just as important, these mutations have been used as a reference point in the development of various cytochemical and biochemical techniques for studying receptor activity.

scholarly journals Soya phytoestrogens, genistein and daidzein, decrease apolipoprotein B secretion from HepG2 cells through multiple mechanisms

2002 ◽  
Vol 366 (2) ◽  
pp. 531-539 ◽  
Author(s):  
Nica M. BORRADAILE ◽  
Linda E. de DREU ◽  
Lisa J. WILCOX ◽  
Jane Y. EDWARDS ◽  
Murray W HUFF
Keyword(s):  
Hepg2 Cells ◽  
Cholesterol Synthesis ◽  
Cholesterol Metabolism ◽  
Plasma Cholesterol ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Mrna Levels ◽  
Apo B ◽  
Cellular Cholesterol ◽  
Apob Secretion

Diets containing the soya-derived phytoestrogens, genistein and daidzein, decrease plasma cholesterol in humans and experimental animals. The mechanisms responsible for the hypocholesterolaemic effects of these isoflavones are unknown. The present study was conducted to determine if genistein and daidzein regulate hepatocyte cholesterol metabolism and apolipoprotein (apo) B secretion in cultured human hepatoma (HepG2) cells. ApoB secretion was decreased dose-dependently by up to 63% and 71% by genistein and daidzein (100μM; P<0.0001) respectively. In contrast, no effect on apoAI secretion was observed. Cellular cholesterol synthesis was inhibited 41% by genistein (100μM; P<0.005) and 18% by daidzein (100μM; P<0.05), which was associated with significant increases in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA. Cellular cholesterol esterification was decreased 56% by genistein (100μM; P<0.04) and 29% by daidzein (100μM; P<0.04); however, mRNA levels for acyl-CoA:cholesterol acyltransferase (ACAT) 1 and ACAT2 were unaffected. At 100μM, both isoflavones equally inhibited the activities of both forms of ACAT in cells transfected with either ACAT1 or ACAT2. Genistein (100μM) and daidzein (100μM) significantly decreased the activity of microsomal triacylglycerol transfer protein (MTP) by 30% and 24% respectively, and significantly decreased MTP mRNA levels by 35% and 55%. Both isoflavones increased low-density lipoprotein (LDL)-receptor mRNA levels by 3- to 6-fold (100μM; P<0.03) and significantly increased the binding, uptake and degradation of 125I-labelled LDL, suggesting that enhanced reuptake of newly secreted apoB-containing lipoproteins contributed to the net decrease in apoB secretion. These results indicate that genistein and daidzein inhibit hepatocyte apoB secretion through several mechanisms, including inhibition of cholesterol synthesis and esterification, inhibition of MTP activity and expression and increased expression of the LDL-receptor.

scholarly journals Co-ordinate regulation of low-density-lipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA reductase and synthase gene expression in HepG2 cells

1989 ◽  
Vol 260 (3) ◽  
pp. 731-736 ◽  
Author(s):  
D T Molowa ◽  
G M Cimis
Keyword(s):  
Hepg2 Cells ◽  
Cholesterol Biosynthesis ◽  
Hepatoma Cell ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Hepatoma Cell Line ◽  
Low Density ◽  
Hmg Coa Reductase ◽  
Coa Reductase

Cellular processes responsible for maintaining cholesterol homoeostasis are highly regulated. To determine whether two of these processes, cholesterol biosynthesis and receptor-mediated uptake of low-density lipoprotein (LDL), are co-ordinately regulated in human liver, we employed a human hepatoma cell line (HepG2) and measured the accumulation of mRNA for LDL receptor, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and HMG-CoA synthase under a variety of conditions. Genomic Southern-blot analysis demonstrated that the integrity of these genes is maintained in the transformed cell. Treatment of HepG2 cells with mevalonate, 25-hydroxycholesterol, LDL, lovastatin or miconazole resulted in a similar effect on the accumulation of all three mRNAs at the concentrations tested. The onset of the response to drug, whether repression or induction of mRNA accumulation, occurred after approximately the same period of exposure for each mRNA. We conclude that the expression of the LDL receptor, HMG-CoA reductase and HMG-CoA synthase is co-ordinately regulated in HepG2 cells.

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