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 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.

The regulated degradation of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase reporter construct occurs in the endoplasmic reticulum

1994 ◽  
Vol 107 (9) ◽  
pp. 2635-2642
Author(s):  
L.W. Lecureux ◽  
B.W. Wattenberg
Keyword(s):  
Endoplasmic Reticulum ◽  
Coenzyme A ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Dna Encoding ◽  
Hmg Coa Reductase ◽  
Steady State Levels ◽  
Coa Reductase ◽  
Membrane Anchoring ◽  
Beta Galactosidase

The rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase, is regulated at a number of levels. One important mechanism is regulation of the half-life of the protein by a controlled proteolytic system. This comes about in response to downstream products of the sterol biosynthetic pathway. Little is known about this system, including where in the cell this regulated degradation occurs. HMG CoA reductase resides in the endoplasmic reticulum. To localize the site of regulated degradation of HMG CoA reductase, we used a construct that fuses the N-terminal membrane-anchoring domain of HMG CoA reductase in-frame with beta-galactosidase as a reporter domain (HM-Gal). HM-Gal has previously been shown to reproduce faithfully the degradative properties of native HMG CoA reductase (Chun et al. (1990) J. Biol. Chem. 265, 22004–22010). CHO cells transfected with DNA encoding HM-Gal were exposed to mevalonic acid, which enhances the rate of HMG CoA reductase degradation several fold, and leads to the reduction of the steady state levels of HM-Gal by 80–90%. To accumulate HMG CoA reductase at the site of degradation, cells were simultaneously treated with N-acetyl-leucyl-leucyl-norleucinal (ALLN), which inhibits the protease responsible for reductase degradation. HM-Gal was localized morphologically by immunofluorescence and biochemically by measuring beta-galactosidase activity in Percoll gradients of cellular homogenates. Using either technique HM-Gal localization was indistinguishable from that of ER markers in both control cells and in cells treated to accumulate HMG CoA reductase at the site of degradation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals 3-Hydroxy-3-methylglutaryl coenzyme A reductase localization in rat liver peroxisomes and microsomes of control and cholestyramine-treated animals: quantitative biochemical and immunoelectron microscopical analyses.

1986 ◽  
Vol 103 (3) ◽  
pp. 875-886 ◽  
Author(s):  
G A Keller ◽  
M Pazirandeh ◽  
S Krisans
Keyword(s):  
Rat Liver ◽  
Coenzyme A ◽  
Specific Activity ◽  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Normal Liver ◽  
Hmg Coa Reductase ◽  
The Matrix ◽  
Coa Reductase ◽  
Liver Peroxisomes

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key regulatory enzyme involved in cholesterol biosynthesis, has recently been reported to be present in rat liver peroxisomes (Keller, G.A., M.C. Barton, D.J. Shapiro, and S.J. Singer, 1985, Proc. Natl. Acad. Sci. USA, 82:770-774). Immunoelectron labeling of ultrathin frozen sections of normal liver, using two monoclonal antibodies to purified rat liver microsomal HMG-CoA reductase, indicated that the enzyme is present in the matrix of peroxisomes. This study is a quantitative biochemical and immunoelectron microscopical analysis of HMG-CoA reductase in rat liver peroxisomes and microsomes of normal and cholestyramine-treated animals. Cholestyramine treatment produced a six- to sevenfold increase in the specific activity of peroxisomal HMG-CoA reductase, whereas the microsomal HMG-CoA reductase specific activity increased by about twofold. Using a computer program that calculates optimal linear combinations of marker enzymes, it was determined that between 20 and 30% of the total reductase activity was located in the peroxisomes of cholestyramine-treated animals. Less than 5% of the reductase activity was present in peroxisomes under control conditions. Quantitation of the immunoelectron microscopical data was in excellent agreement with the biochemical results. After cholestyramine treatment there was an eightfold increase in the density of gold particles per peroxisome, and we estimate about a threefold increase in the labeling of the ER.

A Novel NAD+/MWNTs Nanocomposite-Based Electrochemical Biosensor for Measuring Mevalonic Acid

2014 ◽  
Vol 605 ◽  
pp. 388-391 ◽  
Author(s):  
Rungtiva Palangsuntikul ◽  
Saithip Pakapongpan ◽  
Porntip Khownarumit ◽  
Werasak Surareungchai
Keyword(s):  
Electrocatalytic Activity ◽  
Electrochemical Biosensor ◽  
Limit Of Detection ◽  
Reductase Activity ◽  
Mevalonic Acid ◽  
Fast Response ◽  
Good Stability ◽  
Screen Printed Electrode ◽  
Hmg Coa Reductase ◽  
Coa Reductase

A novel, simple and precise electrochemical biosensor, was developed for measuring mevalonic acid (MA) concentration, which is thought to be a good indicator of HMG-CoA reductase activity. This sensor is based on noncovalent-linking NAD+/MWNTs nanocomposite coated on a screen-printed electrode (SPE). The resulting biosensor exhibited excellent electrocatalytic activity, fast response and good stability to MA. At the NAD+/MWNTs-modified SPE, the current is linear with the concentration of MA being within a concentration range from 18.1 to 145 μM with a limit of detection down to 4.25 μM (S/N = 3), and the sensor exhibited a sensitivity of 92.2 μA/mM.

The effects of tunicamycin, mevinolin and mevalonic acid on HMG-CoA reductase activity and nuclear division in the myxomycete Physarum polycephalum

1989 ◽  
Vol 92 (3) ◽  
pp. 341-344
Author(s):  
W. Engstrom ◽  
O. Larsson ◽  
W. Sachsenmaier
Keyword(s):  
Physarum Polycephalum ◽  
Nuclear Division ◽  
Reductase Activity ◽  
Mevalonic Acid ◽  
Minor Effect ◽  
Hmg Coa Reductase ◽  
Rate Limiting Step ◽  
Coa Reductase ◽  
A Minor ◽  
Rate Limiting

The effects of two inhibitors of 3-hydroxy 3-methyl glutaryl-coenzyme A reductase (tunicamycin and mevinolin) on nuclear division in the myxomycete Physarum polycephalum were examined. Tunicamycin exerted a minor effect on division in synchronized cultures, whereas mevinolin delayed the second, third and fourth nuclear divisions with increasing efficiency. Mevinolin also appeared to be the more potent inhibitor of HMG-CoA reductase, which catalyses the rate-limiting step in the biosynthesis of cholesterol and other isoprene derivatives. These effects of mevinolin could be partially reversed by the addition of mevalonate, suggesting that mevinolin exerts its inhibitory effects on Physarum nuclear division by decreasing the activity of HMG-CoA reductase.

Quantification of urinary mevalonic acid as a biomarker of HMG-CoA reductase activity by a novel translational LC–MS/MS method

Bioanalysis ◽  
2014 ◽  
Vol 6 (7) ◽  
pp. 919-933 ◽  
Author(s):  
Alison VM Rodrigues ◽  
James L Maggs ◽  
Stephen J McWilliam ◽  
Munir Pirmohamed ◽  
Muireann Coen ◽  
...  
Keyword(s):  
Reductase Activity ◽  
Mevalonic Acid ◽  
Hmg Coa Reductase ◽  
Coa Reductase

scholarly journals Cholesterol and mevalonic acid are independent requirements for the in vitro proliferation of human bone marrow granulocyte progenitor cells: studies using ML-236B

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 667-671
Author(s):  
PC Hoffman ◽  
CM Richman ◽  
RA Larson ◽  
S Yachnin
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Human Bone ◽  
Hmg Coa Reductase ◽  
Coa Reductase

ML-236B is a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the key regulatory enzyme in the sequence that catalyzes the conversion of acetate to mevalonic acid in cholesterol biosynthesis. This compound caused marked inhibition of human bone marrow granulocyte progenitor cell (CFU-C) proliferation, the 50% inhibitory concentration (IHD50) being 2.0 X 10(6)M. Inhibition of colony formation was reversed by mevalonic acid but not by cholesterol. ML-236B also inhibited DNA synthesis and acetate incorporation into cholesterol in marrow mononuclear cells (IHD50 = 5.6 x 10(6)M and 3.2 x 10(7)M, respectively). No inhibition of mevalonate incorporation into cholesterol was observed. These results differ from those observed with 25-hydroxycholesterol, another inhibitor of HMG CoA reductase. The latter compound also inhibited CFU-C proliferation and cholesterol biosynthesis from acetate; inhibition of colony formation was reversed by cholesterol but not by mevalonic acid. In addition, 25- hydroxycholesterol inhibited cholesterol synthesis from mevalonic acid precursor. We conclude that: (1) ML-236B is a potent inhibitor of CFU-C proliferation, DNA synthesis, and cholesterol biosynthesis from acetate precursor in marrow mononuclear cells; (2) the effects of ML-236B are completely reversed by mevalonic acid but not by cholesterol, suggesting that mevalonic acid per se or one or more of its nonsterol products are critical for cell growth; (3) the inhibitory effects of 25- hydroxycholesterol on CFU-C proliferation and cholesterol biosynthesis are not solely a result of its inhibition of HMG CoA reductase, but are due in part to inhibition of enzymatic steps distal to mevalonic acid in the sterol synthetic pathway; and (4) mevalonic acid and cholesterol are independent requirements for CFU-C proliferation and differentiation in vitro.

scholarly journals Cholesterol and mevalonic acid are independent requirements for the in vitro proliferation of human bone marrow granulocyte progenitor cells: studies using ML-236B

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 667-671 ◽  
Author(s):  
PC Hoffman ◽  
CM Richman ◽  
RA Larson ◽  
S Yachnin
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Human Bone ◽  
Hmg Coa Reductase ◽  
Coa Reductase

Abstract ML-236B is a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the key regulatory enzyme in the sequence that catalyzes the conversion of acetate to mevalonic acid in cholesterol biosynthesis. This compound caused marked inhibition of human bone marrow granulocyte progenitor cell (CFU-C) proliferation, the 50% inhibitory concentration (IHD50) being 2.0 X 10(6)M. Inhibition of colony formation was reversed by mevalonic acid but not by cholesterol. ML-236B also inhibited DNA synthesis and acetate incorporation into cholesterol in marrow mononuclear cells (IHD50 = 5.6 x 10(6)M and 3.2 x 10(7)M, respectively). No inhibition of mevalonate incorporation into cholesterol was observed. These results differ from those observed with 25-hydroxycholesterol, another inhibitor of HMG CoA reductase. The latter compound also inhibited CFU-C proliferation and cholesterol biosynthesis from acetate; inhibition of colony formation was reversed by cholesterol but not by mevalonic acid. In addition, 25- hydroxycholesterol inhibited cholesterol synthesis from mevalonic acid precursor. We conclude that: (1) ML-236B is a potent inhibitor of CFU-C proliferation, DNA synthesis, and cholesterol biosynthesis from acetate precursor in marrow mononuclear cells; (2) the effects of ML-236B are completely reversed by mevalonic acid but not by cholesterol, suggesting that mevalonic acid per se or one or more of its nonsterol products are critical for cell growth; (3) the inhibitory effects of 25- hydroxycholesterol on CFU-C proliferation and cholesterol biosynthesis are not solely a result of its inhibition of HMG CoA reductase, but are due in part to inhibition of enzymatic steps distal to mevalonic acid in the sterol synthetic pathway; and (4) mevalonic acid and cholesterol are independent requirements for CFU-C proliferation and differentiation in vitro.

Regulation of hepatic cholesterol biosynthesis by berberine during hyperhomocysteinemia

2011 ◽  
Vol 300 (3) ◽  
pp. R635-R643 ◽  
Author(s):  
Nan Wu ◽  
Lindsei K. Sarna ◽  
Yaw L. Siow ◽  
Karmin O
Keyword(s):  
Lipid Accumulation ◽  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Cholesterol Homeostasis ◽  
Enzyme Treatment ◽  
Hepatic Cholesterol ◽  
Hmg Coa Reductase ◽  
Hepatic Lipid ◽  
Hepatic Lipid Accumulation ◽  
Coa Reductase

Hyperhomocysteinemia, an elevation of blood homocysteine levels, is a metabolic disorder associated with dysfunction of multiple organs. We previously demonstrated that hyperhomocysteinemia stimulated hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase leading to hepatic lipid accumulation and liver injury. The liver plays an important role in cholesterol biosynthesis and overall homeostasis. HMG-CoA reductase catalyzes the rate-limiting step in cholesterol biosynthesis. Hepatic HMG-CoA reductase is a major target for lowering cholesterol levels in patients with hypercholesterolemia. The aim of the present study was to examine the effect of berberine, a plant-derived alkaloid, on hepatic cholesterol biosynthesis in hyperhomocysteinemic rats and to identify the underlying mechanism. Hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet for 4 wk. HMG-CoA reductase activity was markedly elevated in the liver of hyperhomocysteinemic rats, which was accompanied by hepatic lipid accumulation. Activation of HMG-CoA reductase was caused by an increase in its gene expression and a reduction in its phophorylation (an inactive form of the enzyme). Treatment of hyperhomocysteinemic rats with berberine for 5 days inhibited HMG-CoA reductase activity and reduced hepatic cholesterol content. Such an inhibitory effect was mediated by increased phosphorylation of HMG-CoA reductase. Berberine treatment also improved liver function. These results suggest that berberine regulates hepatic cholesterol biosynthesis via increased phosphorylation of HMG-CoA reductase. Berberine may be therapeutically useful for the management of cholesterol homeostasis.

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