scholarly journals Inhibition by the fungicide fenpropimorph of cholesterol biosynthesis in 3T3 fibroblasts

1988 ◽  
Vol 256 (3) ◽  
pp. 829-834 ◽  
Author(s):  
M F Corio-Costet ◽  
N Gerst ◽  
P Benveniste ◽  
F Schuber
Keyword(s):  
Mammalian Cells ◽  
Higher Plants ◽  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Acetate Incorporation ◽  
3T3 Fibroblasts ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Coa Reductase ◽  
Hydroxymethylglutaryl Coa Reductase

Fenpropimorph (N-[3-(p-t-butylphenyl)-2-methylpropyl]-cis-2,6-dimethylmorpholine), a morpholine fungicide known to be an inhibitor of sterol biosynthesis in fungi and in higher plants, was demonstrated to be an efficient inhibitor of cholesterol biosynthesis in cultured Swiss 3T3 fibroblasts. Treatment of the mammalian cells with fenpropimorph resulted in a dose-dependent inhibition of [14C]acetate incorporation into the C27 sterols [IC50 (concentration causing half-maximal inhibition) = 0.5 microM], which was accompanied by an accumulation of polar sterols and a decrease in cellular hydroxymethylglutaryl-CoA reductase activity. Exposure of the cells to the drug affected cell growth. Analysis of the sterols in the growth-arrested and in the pulse-labelled cells indicate that fenpropimorph has, in the sterol-biosynthetic pathway, target enzymes in mammalian cells different from those in the other phyla. Whereas in plants and fungi fenpropimorph mainly affects sterol isomerases and reductases, in the fibroblasts its main target seems to be the demethylation of lanosterol.

Treatment of mammalian cells with the endoplasmic reticulum-proliferator compactin strongly induces recombinant and endogenous xenobiotic metabolizing enzymes and 3-hydroxy-3-methylglutaryl-CoA reductase in vitro

1999 ◽  
Vol 112 (4) ◽  
pp. 515-523
Author(s):  
L. McLaughlin ◽  
B. Burchell ◽  
M. Pritchard ◽  
C.R. Wolf ◽  
T. Friedberg
Keyword(s):  
Endoplasmic Reticulum ◽  
Enzyme Activity ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Metabolizing Enzymes ◽  
P450 Reductase ◽  
Coa Reductase

Some xenobiotics induce membrane-bound drug metabolizing enzymes (Xme) and a profound proliferation of the endoplasmic reticulum (ER) in vivo. However these effects are much weaker in vitro, possibly due to absence of certain transcription factors. We tested the possibility that ER proliferation can affect the level of ER-resident enzymes even in the absence of transcriptional activation. For this purpose we analysed the effects of compactin, which has been shown to induce ER proliferation in vitro, on recombinant Xme, which were expressed from a constitutive viral promoter. High levels of recombinant UDP-glucuronosyltransferase UGT1A6 were achieved by amplification of the UGT1A6 cDNA using the dihydrofolate reductase cDNA as selectable marker in DHFR- CHO cells. Treatment of the resulting cell lines with lipoprotein-deficient serum in the absence and presence of compactin for 5 days resulted in a 1.3- and 2.3-fold, respectively, increase of the UGT enzyme activity towards 4-methylumbelliferone, paralleled by an induction of immunoreactive UGT1A6 protein. Similarly, treatment with this 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor increased the endogenous P450 reductase activity 2.6-fold, concomitant with an increase of immunodetectable protein. As expected compactin induced the level of 3-hydroxy-3-methylglutaryl-CoA reductase. Increased levels of this protein have been associated with a proliferation of the ER. Compactin treatment of a separate cell line that expressed recombinant human P450 reductase increased this enzyme activity fivefold. Pulse-chase experiments revealed that the induction of the recombinant Xme by compactin was most likely due to decreased protein degradation. Our results show that enzyme systems unrelated to those involved in cholesterol biosynthesis are affected by compounds known to affect membrane biogenesis. Since this effect extends to heterologously expressed enzymes, it also provides an efficient means by which to increase the levels of recombinant ER proteins.

scholarly journals The role of substrate supply in the regulation of cholesterol biosynthesis in rat hepatocytes

1983 ◽  
Vol 210 (3) ◽  
pp. 625-632 ◽  
Author(s):  
C R Pullinger ◽  
G F Gibbons
Keyword(s):  
Biosynthetic Pathway ◽  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Rat Hepatocytes ◽  
Sterol Synthesis ◽  
Hmg Coa Reductase ◽  
Coa Reductase ◽  
Opposing Effects ◽  
Hydroxymethylglutaryl Coa Reductase

1. Compactin, (-)-hydroxycitrate and dexamethasone gave rise to a decrease in the rate of cholesterol production in hepatocytes from fed rats by interfering with the flow of substrate into the sterol biosynthetic pathway. The cells responded to the deficit of biosynthetic sterol by increasing the activity of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase). 2. Compactin and (-)-hydroxycitrate gave similar results in hepatocytes from rats starved for 24 h but in this case dexamethasone had no significant effect. 3. Exogenous oleate interferes with the production of carbohydrate-derived acetyl-CoA and also gives rise initially to opposing effects on the rate of sterol synthesis and HMG-CoA reductase activity. Over a longer period, however, oleate itself was capable of replacing carbohydrate as the major source of carbon for sterol synthesis. 4. The increase in HMG-CoA reductase activity observed when liver cells were incubated in the presence of compactin, (-)-hydroxycitrate or oleate could be partially reversed by the simultaneous presence of glucagon. 5. Under some physiological conditions, a deficiency of biosynthetic cholesterol or of a related precursor may lead to an increase in the activity of HMG-CoA reductase.

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

1984 ◽  
Vol 219 (2) ◽  
pp. 461-470 ◽  
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.

scholarly journals Regulation of hepatic cholesterol biosynthesis. Effects of a cytochrome P-450 inhibitor on the formation and metabolism of oxygenated sterol products of lanosterol

1989 ◽  
Vol 264 (2) ◽  
pp. 495-502 ◽  
Author(s):  
J Iglesias ◽  
G F Gibbons
Keyword(s):  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Subcellular Fractions ◽  
Hmg Coa Reductase ◽  
Hepatocyte Cultures ◽  
C 32 ◽  
Lanosterol Demethylase ◽  
Cholesterol Precursor ◽  
Coa Reductase

The involvement of oxygenated cholesterol precursors in the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was studied by examining the effect of ketoconazole on the metabolism of mevalonic acid, lanosterol and the lanosterol metabolites, lanost-8-ene-3 beta,32-diol,3 beta-hydroxylanost-8-en-32-al and 4,4-dimethylcholesta-8,14-dien-3 beta-ol, in liver subcellular fractions and hepatocyte cultures. Inhibition of cholesterol synthesis from mevalonate by ketoconazole at concentrations up to 30 microM was due exclusively to a suppression of cytochrome P-450LDM (LDM = lanosterol demethylase) activity, resulting in a decreased rate of lanosterol 14 alpha-demethylation. No enzyme after the 14 alpha-demethylase step was affected. When [14C]mevalonate was the cholesterol precursor, inhibition of cytochrome P450LDM was accompanied by the accumulation of several labelled oxygenated sterols, quantitatively the most important of which was the C-32 aldehyde derivative of lanosterol. There was no accumulation of the 24,25-oxide derivative of lanosterol, nor of the C-32 alcohol. Under these conditions the activity of HMG-CoA reductase declined. The C-32 aldehyde accumulated to a far greater extent when lanost-8-ene-3 beta,32-diol rather than mevalonate was used as the cholesterol precursor in the presence of ketoconazole. With both precursors, this accumulation was reversed at higher concentrations of ketoconazole in liver subcellular fractions. A similar reversal was not observed in hepatocyte cultures.

scholarly journals Identification of Metal Dithiocarbamates as a Novel Class of Antileishmanial Agents

2015 ◽  
Vol 59 (4) ◽  
pp. 2144-2152 ◽  
Author(s):  
Dhiman Sankar Pal ◽  
Dipon Kumar Mondal ◽  
Rupak Datta
Keyword(s):  
Mammalian Cells ◽  
Leishmania Major ◽  
Lethal Dose ◽  
Parasite Burden ◽  
Intracellular Acidosis ◽  
Content Type ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Metal Dithiocarbamates ◽  
Morphological Deformities

ABSTRACTDithiocarbamates have emerged as potent carbonic anhydrase (CA) inhibitors in recent years. Given that CAs are important players in cellular metabolism, the objective of this work was to exploit the CA-inhibitory property of dithiocarbamates as a chemotherapeutic weapon against theLeishmaniaparasite. We report here strong antileishmanial activity of three hitherto unexplored metal dithiocarbamates, maneb, zineb, and propineb. They inhibited CA activity inLeishmania majorpromastigotes at submicromolar concentrations and resulted in a dose-dependent inhibition of parasite growth. Treatment with maneb, zineb, and propineb caused morphological deformities of the parasite andLeishmaniacell death with 50% lethal dose (LD50) values of 0.56 μM, 0.61 μM, and 0.27 μM, respectively. These compounds were even more effective against parasites growing in acidic medium, in which their LD50values were severalfold lower. Intracellular acidosis leading to apoptotic and necrotic death ofL. majorpromastigotes was found to be the basis of their leishmanicidal activity. Maneb, zineb, and propineb also efficiently reduced the intracellular parasite burden, suggesting that amastigote forms of the parasite are also susceptible to these metal dithiocarbamates. Interestingly, mammalian cells were unaffected by these compounds even at concentrations which are severalfold higher than their antileishmanial LD50s). Our data thus establish maneb, zineb, and propineb as a new class of antileishmanial compounds having broad therapeutic indices.

scholarly journals Activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in brains of adult and 7-day-old rats

1976 ◽  
Vol 154 (2) ◽  
pp. 559-560 ◽  
Author(s):  
M M. Sudjic ◽  
R Booth
Keyword(s):  
Rat Brain ◽  
Coenzyme A ◽  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Adult Brain ◽  
Adult Rat ◽  
Adult Rat Brain ◽  
Old Rats ◽  
Coa Reductase ◽  
Coenzyme A Reductase

Rat brain contains 3-hydroxy-3-methylglutaryl-CoA reductase activity, but this enzyme is far more active in 7-day-old brain than in adult brain. This difference may partly explain why cholesterol biosynthesis is more rapid in growing than in adult rat brain.

scholarly journals A discoordinate increase in the cellular amount of 3-hydroxy-3-methylglutaryl-CoA reductase results in the loss of rate-limiting control over cholesterogenesis in a tumour cell-free system

1989 ◽  
Vol 258 (2) ◽  
pp. 421-425 ◽  
Author(s):  
N I Azrolan ◽  
P S Coleman
Keyword(s):  
Cholesterol Biosynthesis ◽  
Normal Liver ◽  
Preliminary Evidence ◽  
Free System ◽  
Normal Rat ◽  
Morris Hepatoma ◽  
Coa Reductase ◽  
Rate Limiting ◽  
Hydroxymethylglutaryl Coa Reductase ◽  
Tumour System

Cholesterol biosynthesis was characterized in cell-free post-mitochondrial supernatant systems prepared from both normal rat liver and Morris hepatoma 3924A. The rate of cholesterol synthesis per cell was 9-fold greater in the tumour system than in that from normal liver, and the tumour systems showed the loss of rate-limiting control at the hydroxymethylglutaryl-CoA reductase (HMGR)-catalysed step. The apparent absence of rate-limiting control over cell-free tumour cholesterogenesis was traced primarily to a discoordinate and dramatic increase in the amount of HMGR in the tumour relative to the liver system. Preliminary evidence for an altered control of the post-lanosterol portion of the pathway was also obtained with the tumour system.

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