Plasma membrane sphingomyelin and the regulation of HMG-CoA reductase activity and cholesterol biosynthesis in cell cultures

We have examined the mechanism of the inhibition of cholesterol synthesis in cells treated with exogenous sphingomyelinase. Treatment of rat intestinal epithelial cells (IEC-6), human skin fibroblasts (GM-43), and human hepatoma (HepG2) cells in culture with sphingomyelinase resulted in a concentration- and time-dependent inhibition of the activity of HMG-CoA reductase, a key regulatory enzyme in cholesterol biosynthesis. The following observations were obtained with IEC-6 cells. Free fatty acid synthesis or general cellular protein synthesis was unaffected by the addition of sphingomyelinase. Addition of sphingomyelinase to the in vitro reductase assay had no effect on activity, suggesting that an intact cell system is required for the action of sphingomyelinase. The products of sphingomyelin hydrolysis, e.g., ceramide and phosphocholine, had no effect on reductase activity. Sphingosine, a further product of ceramide metabolism, caused a stimulation of reductase activity. Examination of the incorporation of [3H]acetate into the nonsaponifiable lipid fractions in the presence of sphingomyelinase showed no changes in the percent distribution of radioactivity in the post-mevalonate intermediates of the cholesterol biosynthetic pathway, but there was increased radioactivity associated with the polar sterol fraction. Pretreatment of cells with ketoconazole, a known inhibitor of oxysterol formation, prevented the inhibition of reductase activity by sphingomyelinase and decreased the incorporation of [3H]acetate in the polar sterol fraction. Ketoconazole had no effect on exogenous sphingomyelinase activity in vitro in the presence or absence of cells. Endogenous sphingomyelinase activity was also unaffected by ketoconazole. Addition of inhibitors of endogenous sphingomyelinase activity, e.g., chlorpromazine, desipramine, and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), to the culture medium caused a dose-dependent stimulation of reductase activity. However, these agents had no effect on the inhibition of reductase activity by exogenous sphingomyelinase. Treatment of cells with small unilamellar vesicles of dioleyl phosphatidylcholine or high density lipoprotein3 resulted in increased efflux of cholesterol and stimulation of reductase activity. Under similar conditions, the inhibitory effect of exogenous sphingomyelinase on reductase activity was prevented by incubation with small unilamellar vesicles of phosphatidylcholine or high density lipoprotein. These results support the hypothesis that alteration of the ratio of sphingomyelin:cholesterol in the plasma membrane plays a modulatory role on the flow of membrane cholesterol to a site where it may be converted to a putative regulatory molecule, possibly an oxysterol.     

Rapid reverse phase-HPLC assay of HMG-CoA reductase activity

Radioisotope-based and mass spectrometry coupled to chromatographic techniques are the conventional methods for monitoring HMG-CoA reductase (HMGR) activity. Irrespective of offering adequate sensitivity, these methods are often cumbersome and time-consuming, requiring the handling of radiolabeled chemicals or elaborate ad-hoc derivatizing procedures. We propose a rapid and versatile reverse phase-HPLC method for assaying HMGR activity capable of monitoring the levels of both substrates (HMG-CoA and NADPH) and products (CoA, mevalonate, and NADP⁺) in a single 20 min run with no pretreatment required. The linear dynamic range was 10–26 pmol for HMG-CoA, 7–27 nmol for NADPH, 0.5–40 pmol for CoA and mevalonate, and 2–27 nmol for NADP⁺, and limit of detection values were 2.67 pmol, 2.77 nmol, 0.27 pmol, and 1.3 nmol, respectively.HMG-CoA reductase (HMGR) is the enzyme that catalyze the four-electron reductive deacylation of HMG-CoA to CoA and mevalonate (Fig. 1) (1). This reaction is the controlling step in the biosynthesis of sterols and isoprenoids (2, 3); hence, a large number of studies on the modulation of HMGR activity are continuously performed in the effort of developing new drugs in the treatment of hypercholesterolemic disorders (1).Open in a separate windowFig. 1.Schematic representation of HMGR enzymatic reaction.HMGR activity is conventionally assayed using elaborate radiochemical assay (4–9), chromatographic techniques coupled with mass spectrometry (10–15), or spectrophotometrically by monitoring the decrease in the absorbance of cofactor NADPH at 340 nm (16).Herein, as an alternative for laboratories with no access to the expensive LC/MS equipment, we propose a rapid and adequately sensitive HPLC-based method capable of monitoring both the levels of all the species involved in the equilibrium in a single analysis and the kinetics of HMGR-catalyzed reactions.     

Measurement of human leukocyte microsomal HMG-CoA reductase activity

Methods were developed for determination of microsomal HMG-CoA reductase activity from freshly isolated human lymphocytes, monocytes, and granulocytes or cultured human lymphoid cells. Reductase activity in monocytes is approximately twice that in lymphocytes or granulocytes. The activity in cultured cells is approximately 34-fold greater than that in freshly isolated cells. Assay conditions were such as to preclude formation of HMG-CoA cleavage products. Leukocyte reductase activity was inhibited by dichloroacetate, a noncompetitive inhibitor of rat liver reductase and a serum cholesterol-lowering agent in man. Measurement of microsomal reductase activity from freshly isolated leukocytes may prove useful in assessing in vivo regulation of cholesterol synthesis in man.     

HMG-CoA reductase kinase: measurement of activity by methods that preclude interference by inhibitors of HMG-CoA reductase activity or by mevalonate kinase

Assay of HMG-CoA reductase kinase activity requires HMG-CoA reductase (reductase, E.C. 1.1.1.34) free of associated reductase kinase. Microsomal reductase insensitive to inactivation by Mg-nucleotides alone may be prepared by heating microsomes at 50 degrees C for 15 min. The reductase in these microsomes may subsequently be inactivated by Mg-nucleotides only after addition of reductase kinase. Inactivation is a linear function of time and of cytosol protein concentration and may be reversed by treatment with a phosphoprotein phosphatase. The extent of inactivation observed under standard conditions provides an assay for reductase kinase activity. Factors present in cytosol that hinder measurement of either reductase or reductase kinase activity must be removed or inhibited. Reductase phosphatase is inhibited by 50 mM NaF. Reductase kinase kinase activity is not expressed under the assay conditions used. Mg-Nucleotide-independent inhibitors of reductase activity are removed by chromatography on DEAE-Sephacel or Blue Sepharose. Mevalonate kinase and reductase kinase are separable by chromatography on DEAE-Sephacel or Sephadex G-200. We describe a rapid chromatographic procedure for separating reductase kinase of crude fractions from mevalonate kinase and from Mg-nucleotide-independent inhibitors of reductase activity. The 1.0 M KCl eluate from DEAE-Sephacel contains all of the cytosol reductase kinase activity. This method is applicable to measurement of reductase kinase activity in cytosol or more purified fractions.     

Modulation of HMG-CoA reductase activity by pantetheine/pantethine

The ability of pantetheine/pantethine to modulate the activity of HMG-CoA reductase (EC 1.1.1.34) was determined in vitro with rat liver microsomes. The decay of the activity was obtained with pantethine in the 10(-5)-10(-4) M range, whereas stimulation by pantetheine occurred at 10(-3)-10(-2) M, as previously reported for GSSG and GSH, respectively. Inhibition of HMG-CoA by pantethine in isolated liver cells was also investigated by measuring the enzyme activity in microsomes isolated from hepatocytes incubated without or with 1 mM pantethine under conditions previously shown by us to induce inhibition of cholesterol synthesis from acetate. The enzyme amount was not modified by pantethine, but in cells treated with the disulphide, the relative amounts of the thiolic active forms of the enzyme, both phosphorylated and dephosphorylated, were decreased to about half compared to controls.     

In situ measurement of HMG-CoA reductase activity in digitonin-permeabilized hepatocytes

An assay procedure for HMG-CoA reductase is described which allows rapid measurement of the activity of this enzyme in isolated rat hepatocytes. In a one step procedure digitonin permeabilizes the plasma membrane and at the same time HMG-CoA reductase activity is measured. Digitonin at a concentration of 64 micrograms per mg of cell protein was found to be optimal for exposing microsomal HMG-CoA reductase to the assay components. The enzyme assay is linear with time up til 5 min and with protein concentrations in the range of 0.06-0.6 mg of cell protein per assay. It is shown that cellular enzyme activity is affected by preincubation of intact hepatocytes with a variety of short-term modulators of hepatic cholesterogenesis.     

Diurnal variation of HMG-CoA reductase activity in rat liver peroxisomes

The specific activity of hepatic microsomal and peroxisomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) was determined at different times during a 24 hour cycle from cholestyramine treated rats. The microsomal HMG-CoA reductase activity displayed a peak at D-6 (6th hour of the dark cycle) as previously reported, whereas, the peroxisomal HMG-CoA reductase activity was the highest at L-2 (2nd hour of the light cycle). Immunoblots of the peroxisomal HMG-CoA reductase suggest that the increase in enzyme activity at L-2 is due to changes in enzyme mass. The different cyclic variations observed in microsomal and peroxisomal HMG-CoA reductase activity may suggest different mechanisms of regulation.     

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

Treatment of rat intestinal epithelial cells (IEC-6 cells) with lanosterol 14 alpha-demethylase inhibitors, ketoconazole and miconazole, had similar effects on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and cholesterol biosynthesis but the drugs differed in their ability to prevent the low density lipoprotein (LDL) suppression of reductase activity. Miconazole, at concentrations that inhibited the metabolism of lanosterol and epoxylanosterol to the same degree as ketoconazole, did not prevent low density lipoprotein action on reductase activity, whereas ketoconazole totally abolished the low density lipoprotein action on reductase activity. Both drugs caused: 1) a biphasic response in reductase activity such that at low concentrations (less than 2 microM) reductase activity was inhibited and at high concentrations (greater than 5 microM) the activity returned to control or higher than control levels; 2) an inhibition of metabolism of lanosterol to cholesterol, and 24(S), 25-epoxylanosterol to 24(S), 25-epoxycholesterol. Neither drug prevented suppression of reductase activity by 25-hydroxylanosterol, 25-hydroxycholesterol, or mevalonolactone added to the medium. Each drug increased the binding, uptake, and degradation of 125I-labeled LDL and inhibited the re-esterification of free cholesterol to cholesteryl oleate and cholesteryl palmitate. The release of free cholesterol from [3H]cholesteryl linoleate LDL could not account for the differential effect of ketoconazole and miconazole on the prevention of low density lipoprotein suppression of reductase activity. The differential effect of the drugs on low density lipoprotein suppression of reductase activity was not unique to IEC-6 cells, but was also observed in several cell lines of different tissue origin such as human skin fibroblast cells (GM-43), human hepatoblastoma cells (HepG2), and Chinese hamster ovary cells (wild type, K-1; 4 alpha-methyl sterol oxidase mutant, 215). These observations suggest that the suppressive action of low density lipoprotein on reductase activity 1) does not require the de novo synthesis of cholesterol, or 24(S), 25-epoxysterols; 2) is not mediated via the same mechanism as that of mevalonolactone; and 3) does not involve cholesteryl reesterification. Ketoconazole blocks a site in the process of LDL suppression of reductase activity that is not affected by miconazole.     

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and the rate of sterol synthesis diminish in cultures with high cell density

The specific activity of HMG-CoA reductase, the major rate-limiting enzyme in the sterol biosynthetic pathway, declined linearly with increasing cell density in four different lines of mammalian cell cultures. As expected, this caused the rates of sterol synthesis from [14C]acetate to decline in a parallel manner. The decrease in reductase activity in the dense cultures was also correlated with decreased incorporation of [14C]acetate into fatty acids and [3H]thymidine into DNA. In contrast, the activities of two enzymes, NADH dehydrogenase and 5'-nucleotidase, which are not involved in lipid synthesis, were independent of changes in cell density. The simplest explanation for these data is tht HMG-CoA reductase and the synthesis of sterol and fatty acids are regulated in concordance with the rate of cell growth and proliferation.     

The effect of ACTH on HMG-CoA reductase activity in hamster adrenals

Injection to hamsters of various low doses of ACTH resulted in gradual increases in adrenal HMG-CoA reductase activity, in plasma and adrenal corticosteroid concentrations but produced no change in adrenal cholesterol content. These data indicate that under physiological conditions, ACTH could regulate HMG-CoA reductase activity through a mechanism which does no apparently involve a change in the cholesterol content of the gland.     

Highly sensitive assay of HMG-CoA reductase activity by LC-ESI-MS/MS

We have developed a new sensitive and specific nonradioisotope assay method to measure the activity of HMG-CoA reductase, the rate-controlling enzyme in the cholesterol biosynthetic pathway. This method was based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry using electrospray ionization in positive mode. Mevalonic acid, the product of HMG-CoA reductase, was converted to mevalonolactone (MVL) in an incubation mixture, extracted by a salting-out procedure, derivatized into the mevalonyl-(2-pyrrolidin-1-yl-ethyl)-amide, and then purified using a disposable silica cartridge. The resulting mevalonylamide was quantified by selected reaction monitoring using the positive electrospray ionization mode. The detection limit of this mevalonylamide was found to be 240 amol (signal-to-noise ratio=3), approximately 833 times more sensitive than that of MVL measured by a conventional radioisotope (RI) method (200 fmol). The variances between sample preparations and between measurements by this method were analyzed by one-way layout and calculated to be 3.2% and 1.8%, respectively. The recovery experiments were performed using incubation mixtures spiked with 0.77-2.31 nmol MVL/mg protein and were validated by a polynomial equation. These results showed that the estimated concentration within a 95% confidence limit was 0.47+/-0.07 nmol/mg protein, which coincided completely with the observed X0 nmol/mg protein with a mean recovery of 94.6%. This method made it possible to measure HMG-CoA reductase activity with a high degree of reproducibility and reliability, and especially with sensitivity superior to that of the conventional RI method.     

Effectiveness of flashing light for increasing photosynthetic efficiency of microalgal cultures over a critical cell density

Critical cell density (CCD), the maximum cell concentration without mutual shading in algal cultures, can be used as a new operating parameter for high-density algal cultures and for the application of the flashing light effect on illuminated algal cultures. CCD is a function of average cell volume and light illumination area. The CCD is thus proposed as an index of estimation of mutual shading in algal cultures. Where cell densities are below the CCD, all the cells in photobio-reactors can undergo photosysnthesis at their maximum rate. At cell densities over the CCD, mutual shading will occur and some cells in the illumination chamber cannot grow photoautotrophically. When the cell concentration is higher than the CCD, specific oxygen production rates under flashing light were higher than those under continuous light. The CCD was found to be a useful engineering parameter for the application of flashing light, particularly in high-density algal cultures.     

Relationships between nitrite reductase activity and genotype-dependent callus growth in rice cell cultures

The nitrite ion content and activity of nitrate reductase and nitrite reductase were examined in scutellum-derived calluses of rice varieties using a modified R2 medium (medium A) and a medium derived from the modified R2 medium (medium B). In medium A, marked differences were observed in callus growth between the varieties. The calluses of the poor-growth varieties accumulated significantly more nitrite ions during the culture period than did the good-growth varieties. Callus growth rate was negatively correlated with the nitrite ion content, indicating that the calluses of the poor-growth varieties were injured by toxic nitrite ions, which lead to browning and inhibited growth. The calluses of the poor-growth varieties had significantly lower levels of nitrite reductase activity than good-growth varieties. On the other hand, no between-group differences were observed in the nitrate reductase activity. These results indicate that the higher nitrite ion levels observed in the poor-growth varieties resulted from a lower ability to reduce nitrite and that nitrite reductase activity is one of the physiological factors that correlates with differences between varieties in rice cell cultures. In medium B, the calluses of the poor-growth varieties grew as well as the good-growth varieties, but also had significantly lower levels of nitrite reductase. Nitrate reductase activity was repressed in the calluses of both varieties in medium B compared to culture in medium A. The results suggest that repressed nitrate reductase activity causes the calluses of poor-growth varieties to accumulate only trace amounts of nitrite ions despite lower nitrite reductase activity and as a result, callus growth improved in medium B. Received: 28 July 1998 / Revision received: 14 October 1998 / Accepted: 27 October 1998     

Supernatant protein factor stimulates HMG-CoA reductase in cell culture and in vitro

Supernatant protein factor (SPF) is a 46-kDa cytosolic protein that stimulates squalene monooxygenase in vitro and, unexpectedly, cholesterol synthesis in cell culture. Because squalene monooxygenase is not thought to be rate-limiting with regard to cholesterol synthesis, we investigated the possibility that SPF might stimulate other enzymes in the cholesterol biosynthetic pathway. Substitution of [(14)C]mevalonate for [(14)C]acetate in McARH7777 hepatoma cells expressing SPF reduced the 1.8-fold increase in cholesterol synthesis by half, suggesting that SPF acted on or prior to mevalonate synthesis. This conclusion was supported by the finding that substitution with [(14)C]mevalonate completely blocked an SPF-induced increase in squalene synthesis. Evaluation of 2,3-oxidosqualene synthesis from [(14)C]mevalonate demonstrated that SPF also stimulated squalene monooxygenase (1.3-fold) in hepatoma cells. Immunoblot analysis showed that SPF did not increase HMG-CoA reductase or squalene monooxygenase enzyme levels, indicating a direct effect on enzyme activity. Addition of purified recombinant SPF to rat liver microsomes stimulated HMG-CoA reductase by about 1.5-fold, and the SPF-concentration/activation curve paralleled that for the SPF-mediated stimulation of squalene monooxygenase. These results reveal that SPF directly stimulates HMG-CoA reductase, the rate-limiting step of the cholesterol biosynthetic pathway, as well as squalene monooxygenase, and suggest a new means by which cholesterol synthesis can be rapidly modulated in response to hormonal and environmental signals.     

Binding thermodynamics of statins to HMG-CoA reductase

The statins are powerful inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMG-CoA reductase), the key enzyme in the cholesterol biosynthetic pathway, and are among the most widely prescribed drugs in the world. Despite their clinical importance, little is known about the binding thermodynamics of statins to HMG-CoA reductase. In this paper, we report the results of inhibition kinetics and microcalorimetric analysis of a representative type I statin (pravastatin) and four type II statins (fluvastatin, cerivastatin, atorvastatin, and rosuvastatin). Inhibition constants (K(i)) range from 2 to 250 nM for the different statins. Isothermal titration calorimetry (ITC) experiments yield binding enthalpies (DeltaH(binding)) ranging between zero and -9.3 kcal/mol at 25 degrees C. There is a clear correlation between binding affinity and binding enthalpy: the most powerful statins bind with the strongest enthalpies. The proportion by which the binding enthalpy contributes to the binding affinity is not the same for all statins, indicating that the balance among hydrogen bonding, van der Waals, and hydrophobic interactions is not the same for all of them. At 25 degrees C, the dominant contribution to the binding affinity of fluvastatin, pravastatin, cerivastatin, and atorvastatin is the entropy change. Only for rosuvastatin does the enthalpy change contribute more than 50% of the total binding energy (76%). Since the enthalpic and entropic contributions to binding originate from different types of interactions, the thermodynamic dissection presented here provides a way to identify interactions that are critical for affinity and specificity.     

Activation of HMG-CoA reductase by microsomal phosphatase

HMG-CoA reductase activity can be modulated by a reversible phosphorylation-dephosphorylation with the phosphorylated form of the enzyme being inactive and the dephosphorylated form, active. Phosphatases from diverse sources, including cytosol, have been shown to dephosphorylate and activate HMG-CoA reductase. The present study demonstrates phosphatase activity capable of activating HMG-CoA reductase that is associated with purified microsomes. The incubation of microsomes at 37 degrees C for 40 min results in a twofold stimulation of HMG-CoA reductase activity, and this stimulation is blocked by sodium fluoride or phosphate. The ability of microsomes to increase HMG-CoA reductase activity occurs regardless of whether microsomes are prepared by ultracentrifugation or calcium precipitation. Additionally, phosphatases capable of activating HMG-CoA reductase are present in both the smooth and rough endoplasmic reticulum. Freeze-thawing does not prevent microsomes from activating HMG-CoA reductase but preincubation results in a significant decrease in the ability of microsomes to increase HMG-CoA reductase activity. Thus, the present study demonstrates that purified liver microsomes contain phosphatase activity capable of activating HMG-CoA reductase.     

Intestinal HMG-CoA reductase activity is low in hypercholesterolemic patients and is further decreased with lovastatin therapy

Significant cholesterol synthesis occurs in gut mucosa of animals and humans. However, the role of gut synthesis in hypercholesterolemia and the effect of drugs on this function have not been defined. We obtained jejunal biopsies and bile samples from 21 Type II hypercholesterolemic subjects (mean serum cholesterol = 331 mg/dl) on a low fat diet after an over-night fast. Whole gut mucosal homogenate was assayed for activity of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, the rate-determining enzyme of cholesterol synthesis. Mean reductase activity (pmol/mg per min) was 5.5 +/- 1.0 (n = 21) in hypercholesterolemic subjects versus 11.3 +/- 1.0 in 52 normal subjects (P less than 0.01). This is consistent with the hypothesis that the primary defect in these patients is not excessive cholesterol synthesis but decreased low density lipoprotein (LDL) clearance. It implies that high LDL levels down-regulate gut reductase activity. After treatment of 7 patients with lovastatin (40-80 mg/day for at least 6-13 weeks), gut reductase activity decreased from 7.7 +/- 2.6 to 3.6 +/- 0.5 (P less than 0.05), in biopsies obtained 12 hr after the last drug dose. Inhibition of reductase activity by this drug was detected 12 hr after a dose, and the enzyme was not measurably induced during 6-13 weeks of therapy. In keeping with the decrease in serum cholesterol (332----239 mg/dl) and mucosal reductase activity during lovastatin therapy, mean gallbladder bile cholesterol saturation index also decreased (1.045 +/- 0.112 vs. 0.883 +/- 0.109, n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)