Differential regulation of low density lipoprotein suppression of HMG-CoA reductase activity in cultured cells by inhibitors of cholesterol biosynthesis.

Compactin, an inhibitor of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase, decreased cholesterol synthesis in intact Hep G2 cells. However, after the inhibitor was washed away, the HMG-CoA-reductase activity determined in the cell homogenate was found to be increased. Also the high-affinity association of LDL (low-density lipoprotein) to Hep G2 cells was elevated after incubation with compactin. Lipoprotein-depleted serum, present in the incubation medium, potentiated the compactin effect compared with incubation in the presence of human serum albumin. Addition of either mevalonate or LDL prevented the compactin-induced rise in activities of both HMG-CoA reductase and LDL receptor in a comparable manner. It is concluded that in this human hepatoma cell line, as in non-transformed cells, both endogenous mevalonate or mevalonate-derived products and exogenous cholesterol are able to modulate the HMG-CoA reductase activity as well as the LDL-receptor activity.

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Modulation of regulatory oxysterol formation and low density lipoprotein suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity by ketoconazole. A role for cytochrome P-450 in the regulation of HMG-CoA reductase in rat intestinal epithelial cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)83919-4 ◽

1986 ◽

Vol 261 (18) ◽

pp. 8348-8356 ◽

Cited By ~ 16

Author(s):

A Gupta ◽

R C Sexton ◽

H Rudney

Keyword(s):

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Reductase Activity ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Low Density ◽

Intestinal Epithelial ◽

Hmg Coa Reductase ◽

Coa Reductase ◽

Cytochrome P 450

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

Biochemical Journal ◽

10.1042/bj2600731 ◽

1989 ◽

Vol 260 (3) ◽

pp. 731-736 ◽

Cited By ~ 41

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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Regulation of low density lipoprotein receptor and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities are differentially affected in Niemann–Pick type C and type D fibroblasts

Biochemistry and Cell Biology ◽

10.1139/o93-069 ◽

1993 ◽

Vol 71 (9-10) ◽

pp. 467-474 ◽

Cited By ~ 9

Author(s):

Harkirat S. Sidhu ◽

Stella A. R. Rastogi ◽

David M. Byers ◽

Duane L. Guernsey ◽

Harold W. Cook ◽

...

Keyword(s):

Reductase Activity ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Cell Types ◽

Low Density ◽

Hmg Coa Reductase ◽

Type D ◽

Density Lipoprotein Receptor ◽

Niemann Pick ◽

Coa Reductase

Defective regulation of intracellular cholesterol metabolism has been investigated in cultured fibroblasts from two subtypes of Niemann–Pick type II disease: the panethnic Niemann–Pick type C (NPC) and the Nova Scotia type D (NPD). Cell extracts from NPC and NPD fibroblasts cultured in lipoprotein-deficient medium exhibited activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase that were two-fold greater than in normal cells. Addition of serum resulted in only a 15% decrease in HMG-CoA reductase activity within 6 h in these cells, compared with a decrease of 80% in normal fibroblasts. The initial rate of return to maximal values for the first 6 h after removal of serum was similar in all three cell types; thereafter, the rate was faster in the mutant fibroblasts. Binding and internalization of 125I-labeled low density lipoprotein (LDL) was not decreased within 12 h of incubation of NPC fibroblasts with serum, while a decrease of 50% was observed for both NPD and normal fibroblasts over this time period. Northern blot analysis also indicated a slower decrease in steady-state LDL receptor mRNA in NPC relative to normal and NPD cells. In all three cell types, inhibition of HMG-CoA reductase with mevinolin had no effect on serum-stimulated cholesterol esterification, while inhibition of acyl-CoA:cholesterol acyltransferase with Sandoz 58-035 did not influence HMG-CoA reductase activity, indicating that defects in these regulatory mechanisms are independent. Together with previous observations that NPC and NPD fibroblasts exhibit different levels of cholesterol accumulation and esterification, our results suggest that NPD may result from a distinct mutation in a Niemann–Pick type II gene, in which different mutations can differentially alter the various mechanisms of cholesterol regulation.Key words: Niemann–Pick disease, cholesterol regulation, low density lipoprotein receptor, 3-hydroxy-3-methylglutaryl-CoA reductase, acyl-CoA:cholesterol acyltransferase.

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The regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity, cholesterol esterification and the expression of low-density lipoprotein receptors in cultured monocyte-derived macrophages

Biochemical Journal ◽

10.1042/bj2100523 ◽

1983 ◽

Vol 210 (2) ◽

pp. 523-532 ◽

Cited By ~ 24

Author(s):

B L Knight ◽

D D Patel ◽

A K Soutar

Keyword(s):

Reductase Activity ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Low Density ◽

Cholesteryl Esters ◽

Acetate Incorporation ◽

Normal Cells ◽

Hmg Coa Reductase ◽

Ldl Receptors ◽

Coa Reductase

Human blood monocytes cultured in medium containing 20% whole serum showed the greatest activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and [14C]acetate incorporation into non-saponifiable lipids around the 7th day after seeding, the period of greatest growth. Although there was enough low-density lipoprotein (LDL) in the medium to saturate the LDL receptors that were expressed by normal cells at that time, HMG-CoA reductase activity and acetate incorporation were as high in normal cells as in cells from familial-hypercholesterolaemic (FH) patients. Both the addition of extra LDL, which interacted with the cells by non-saturable processes, and receptor-mediated uptake of acetylated LDL significantly reduced reductase activity and increased incorporation of [14C]oleate into cholesteryl esters in normal cells and cells from FH patients (‘FH cells’), and reduced the expression of LDL receptors in normal cells. Pre-incubation for 20h in lipoprotein-deficient medium apparently increased the number of LDL receptors expressed by normal cells but reduced the activity of HMG-CoA reductase in both normal and FH cells. During subsequent incubations the same rate of degradation of acetylated LDL and of non-saturable degradation of LDL by FH cells was associated with the same reduction in HMG-CoA reductase activity, although LDL produced a much smaller stimulation of oleate incorporation into cholesteryl esters. In normal cells pre-incubated without lipoproteins, receptor-mediated uptake of LDL could abolish reductase activity and the expression of LDL receptors. The results suggested that in these cells, receptor-mediated uptake of LDL might have a greater effect on reductase activity and LDL receptors than the equivalent uptake of acetylated LDL. It is proposed that endogenous synthesis is an important source of cholesterol for growth of normal cells, and that the site at which cholesterol is deposited in the cells may determine the nature and extent of the metabolic events that follow.

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The absolute rate of cholesterol biosynthesis in monocyte-macrophages from normal and familial hypercholesterolaemic subjects

Biochemical Journal ◽

10.1042/bj2190461 ◽

1984 ◽

Vol 219 (2) ◽

pp. 461-470 ◽

Cited By ~ 17

Author(s):

D D Patel ◽

C R Pullinger ◽

B L Knight

Keyword(s):

Cholesterol Synthesis ◽

Reductase Activity ◽

Cholesterol Biosynthesis ◽

Specific Radioactivity ◽

Density Lipoprotein ◽

Cellular Protein ◽

True Rate ◽

Hmg Coa Reductase ◽

Coa Reductase ◽

In Cells

The true rate of cholesterogenesis in cultured monocyte-macrophages was determined from the incorporation of [2-14C]acetate into cholesterol, using the desmosterol (cholesta-5,24-dien-3 beta-ol) that accumulated in the presence of the drug triparanol to estimate the specific radioactivity of the newly formed sterols. It was shown that this procedure could be successfully adapted for use with cultured monocytes despite the accumulation of other unidentified biosynthetic intermediates. In cells maintained in 20% (v/v) whole serum approx. 25% of the sterol carbon was derived from exogenous acetate. Cholesterol synthesis was as high in normal cells as in cells from homozygous familial hypercholesterolaemic (FH) subjects and accounted for 50% of the increase in cellular cholesterol. The addition of extra low-density lipoprotein (LDL) reduced cholesterol synthesis, apparently through a decrease in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). When incubated in lipoprotein-deficient serum some cells did not survive, but those that remained showed a normal increase in protein content; the amount of cellular protein and cholesterol in each well did not increase and cholesterol synthesis was reduced by over 80%. HMG-CoA reductase activity fell less dramatically and the proportion of sterol carbon derived from exogenous acetate increased, suggesting that the low rate of cholesterogenesis with lipoprotein-deficient serum was due to a shortage of substrate. The results indicate that under normal conditions monocyte-macrophages obtain cholesterol from endogenous synthesis rather than through receptor-mediated uptake of LDL, and that synthesis together with non-saturable uptake of LDL provides the majority of the cholesterol required to support growth.

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The effect of (−)-hydroxycitrate on the activity of the low-density-lipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA reductase levels in the human hepatoma cell line Hep G2

Biochemical Journal ◽

10.1042/bj2720181 ◽

1990 ◽

Vol 272 (1) ◽

pp. 181-186 ◽

Cited By ~ 44

Author(s):

T A Berkhout ◽

L M Havekes ◽

N J Pearce ◽

P H E Groot

Keyword(s):

Low Density Lipoprotein ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Low Density ◽

Hep G2 ◽

Atp Citrate Lyase ◽

Hmg Coa Reductase ◽

Synthetic Pathway ◽

Citrate Lyase ◽

Coa Reductase

(-)-Hydroxycitrate, a potent inhibitor of ATP citrate-lyase, was tested in Hep G2 cells for effects on cholesterol homoeostasis. After 2.5 h and 18 h incubations with (-)-hydroxycitrate at concentrations of 0.5 mM or higher, incorporation of [1,5-14C]citrate into fatty acids and cholesterol was strongly inhibited. This most likely reflects an effective inhibition of ATP citrate-lyase. Cholesterol biosynthesis was decreased to 27% of the control value as measured by incorporations from 3H2O, indicating a decreased flux of carbon units through the cholesterol-synthetic pathway. After 18 h preincubation with 2 mM-(-)-hydroxycitrate, the cellular low-density-lipoprotein (LDL) receptor activity was increased by 50%, as determined by the receptor-mediated association and degradation. Measurements of receptor-mediated binding versus LDL concentration suggests that this increase was due to an increase in the numbers of LDL receptors. Simultaneously, enzyme levels of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase as determined by activity measurements increased 30-fold. Our results suggest that the increases in HMG-CoA reductase and the LDL receptor are initiated by the decreased flux of carbon units in the cholesterol-synthetic pathway, owing to inhibition of ATP citratelyase. A similar induction of HMG-CoA reductase and LDL receptor was also found after preincubations of cells with 0.3 microM-mevinolin, suggesting that the underlying mechanism for this induction is identical for both drugs.

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