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.

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.     

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

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.     

Increased sensitivity to dietary cholesterol in diabetic and hypothyroid rats associated with low levels of hepatic HMG-CoA reductase expression

We recently postulated that hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase functions as a cholesterol buffer to protect against the serum and tissue cholesterol raising action of dietary cholesterol. This postulate predicts that diminished basal expression of hepatic HMG-CoA reductase results in increased sensitivity to dietary cholesterol. Because diabetic and hypothyroid animals are known to have markedly reduced hepatic HMG-CoA reductase, these animals were selected as models to test our postulate. When rats were rendered diabetic with streptozotocin, their hepatic HMG-CoA reductase activity decreased from 314 to 22 pmol. min(-1). mg(-1), and their serum cholesterol levels increased slightly. When the diabetic animals were challenged with a diet containing 1% cholesterol, their serum cholesterol levels doubled, and their hepatic reductase activity decreased further to 0.9 pmol. min(-1). mg(-1). Hepatic low-density lipoprotein (LDL) receptor immunoreactive protein levels were unaffected in the diabetic rats whether fed cholesterol-supplemented diets or not. In rats rendered hypothyroid by thyroparathyroidectomy, serum cholesterol levels rose from 100 to 386 mg/dl in response to the 1% cholesterol challenge, whereas HMG-CoA reductase activity dropped from 33.8 to 3.4 pmol. min(-1). mg(-1). Hepatic LDL receptor immunoreactive protein levels decreased only slightly in the hypothyroid rats fed cholesterol-supplemented diets. Taken together, these results show that rats deficient in either insulin or thyroid hormone are extremely sensitive to dietary cholesterol largely due to low basal expression of hepatic HMG-CoA reductase.     

Increased 3-hydroxy-3-methyl-glutaryl coenzyme A reductase activity in a virilizing adrenal carcinoma

HMG-CoA reductase activity was determined on microsomal preparations of an adrenal carcinoma and on a control adrenal obtained from palliative surgery for breast carcinoma. In both tissues we also measured [14C]pyruvate incorporation to study the formation of sterols. The endogenous adrenal content of cholesterol and its esters was quantitated. The content of various steroids was also determined in tissues and media before and after incubations in Krebs-Ringer. The carcinoma had a HMG-CoA reductase activity of 972.0 pmol/mg protein/min vs 13.8 for the control adrenal. The tumor incorporated 4.6 pmol of [14C]pyruvate per mg protein per 90 min into digitonin precipitable sterols compared to 0.5 pmol found for the control gland. Free cholesterol and cholesterol esters in tumoral tissue were 0.09/100 mg and 0.02/100 mg tissue respectively, compared to 0.18 and 2.56 in control tissue. The output of corticosteroids and androgens was very high when calculated for the whole tumor. These results suggest that the carcinoma had acquired a high capacity for de novo synthesis of cholesterol which could have served as substrate for the observed high plasma androgen level.     

Regulation of hepatic cholesterol metabolism in humans: stimulatory effects of cholestyramine on HMG-CoA reductase activity and low density lipoprotein receptor expression in gallstone patients

To characterize the metabolic regulatory response to interruption of the enterohepatic circulation of bile acids, we examined the effects of cholestyramine treatment on the rate-limiting steps in cholesterol biosynthesis (HMG-CoA reductase) and bile acid production (cholesterol 7 alpha-hydroxylase) as well as on the heparin-sensitive binding of low density lipoproteins (LDL) (reflecting LDL receptor expression) in human liver. Altogether, 18 normolipidemic patients with uncomplicated cholesterol gallstone disease were treated with cholestyramine (8 g b.i.d.) for 2-3 weeks prior to cholecystectomy, and another 34 cholesterol gallstone patients served as untreated controls. Cholestyramine treatment stimulated cholesterol 7 alpha-hydroxylase more than sixfold, and increased both HMG-CoA reductase activity (552 +/- 60 pmol/min per mg protein vs 103 +/- 9 pmol/min per mg protein) and LDL receptor expression (6.1 +/- 0.8 ng/mg protein; n = 6 vs 2.2 +/- 0.3 ng/mg protein; n = 7). Moreover, there was a good correlation between HMG-CoA reductase activity and LDL receptor binding (rs = +0.71; n = 13), suggesting a simultaneous stimulatory effect to compensate for the increased hepatic cholesterol catabolism due to bile acid depletion caused by cholestyramine. Further evidence for this assumption was the finding of a significant relationship between cholesterol 7 alpha-hydroxylase activity and both LDL receptor expression (rs = +0.77; n = 13) and HMG-CoA reductase activity (rs = +0.76; n = 46). We conclude that in human liver a parallel stimulation of cholesterol synthesis and LDL receptor expression occurs in response to stimulation of bile acid synthesis.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and lipid metabolism in a concanavalin A-resistant Chinese hamster ovary cell line

Lipid metabolism in a concanavalin A-resistant, glycosylation-defective mutant cell line was investigated by comparing growth properties, lipid composition, and lipid biosynthesis in wild-type (WT), mutant (CR-7), and revertant (RCR-7) cells. In contrast to WT and RCR-7, the mutant was auxotrophic for cholesterol, but mevalonolactone did not restore growth on lipoprotein-deficient medium. The use of R-[2-14C]mevalonolactone revealed that CR-7 was deficient in the conversion of lanosterol to cholesterol. Total lipid and phospholipid content and composition were similar in all three cell lines, but CR-7 displayed subnormal content and biosynthesis of cholesterol and unsaturated fatty acids. The mutant was hypersensitive to compactin and was unable to upregulate either 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity or the binding and internalization of 125I-labeled low-density lipoprotein (LDL) in response to lipoprotein deprivation. HMG-CoA reductase activity in all three cell lines showed similar kinetics and phosphorylation status, and the binding kinetics and degradation of 125I-LDL were also similar, suggesting that CR-7 possesses kinetically normal reductase and LDL binding sites, but is deficient in their coordinate regulation. Tunicamycin (1-2 micrograms/ml) strongly and reversibly suppressed reductase activity in WT and RCR-7. CR-7 was resistant to this inhibitor. In WT cells this suppressive effect was accompanied by inhibition of 3H-labeled mannose incorporation into cellular protein, but 3H-labeled leucine incorporation was unaffected. Immunotitration of HMG-CoA reductase activity in extracts of WT cells, cultured in the presence and absence of tunicamycin, showed that suppression of reductase activity reflected the presence of reduced amounts of reductase protein, implying that glycosylation plays an important role in the coordinate regulation of HMG-CoA reductase activity and LDL binding.     

Circadian rhythm of HMG-CoA reductase and insulin in African green monkeys

HMG-CoA reductase in hepatic microsomes and serum insulin display circadian rhythm in two strains of Cercopethicus aethiops. Grivets develop higher levels of serum cholesterol than vervets fed cholesterol. Males (n = 20/gp) were adapted to a light cycle (7:30 a.m.-8:30 p.m.) for 60 days and fed a non-cholesterol diet at 8:30 a.m. and 2:00 p.m. Vervet acrophase, reductase activity was synchronized to serum insulin units with a specific activity of 1480 pmol/min/mg protein changing by 7.5-fold from nadir to acrophase. The activity profile in grivets was asynchronous to vervet fluctuations with peaks at 12 noon (160 pmol/min/mg) and 6 p.m. (275 pmol/min/mg). Insulin levels also peaked near these times. The 24-h reductase activity was over 4 times greater in vervet than grivet livers. The similar rhythmic patterns of reductase and insulin support the notion that insulin plays an important role in the rhythm of HMG-CoA reductase in primates.     

Abnormal cholesterol metabolism in renal clear cell carcinoma

The clear cell form of renal cell carcinoma is known to derive its histologic appearance from accumulations of glycogen and lipid. We have found that the most consistently stored lipid form is cholesteryl ester. Clear cell cancer tissue contained 8-fold more total cholesterol and 35-fold more esterified cholesterol than found in normal kidney. Cholesteryl ester appeared to be formed intracellularly since it was not membrane-bound and since oleate was the predominant form, as opposed to linoleate in lipoprotein cholesteryl esters. The cholesterol in clear cell tumors did not appear to be a result of excessive synthesis from acetate since HMG-CoA reductase (EC 1.1.1.34) activity was lower in cancer tissue than in normal kidney (2.9 +/- 0.8 vs. 7.2 +/- 1.2 pmol/mg of protein per min). In contrast, intracellular activity of fatty acyl-coenzyme A:cholesterol acyl transferase (ACAT, EC 2.3.1.26) was higher in tumor tissue than in normal kidney (2405 +/- 546 vs. 1326 +/- 301 pmol/mg of protein per 20 min) while cytosolic cholesteryl ester hydrolase activity appeared normal. Cholesteryl ester storage in clear cell renal cancer may be a result of a primary abnormality in ACAT activity or it may be a result of reduced release of free cholesterol (relative to cell content) with a secondary elevation in ACAT activity.     

Hepatic HMG-CoA reductase expression and resistance to dietary cholesterol

The premise that the intrinsic level of expression of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase determines the relative sensitivity to the serum cholesterol raising action of dietary cholesterol was examined in 9 strains of rat. For further comparison purposes, hamsters were also examined. The basal expression of hepatic HMG-CoA reductase, extent of feedback regulation by cholesterol, and changes in serum cholesterol levels and the hepatic low-density lipoprotein (LDL) receptor in response to cholesterol challenge were determined in these animals. The Sprague-Dawley, Wistar-Furth, Spontaneously Hypertensive, Lewis, and Wistar-Kyoto rats were all very resistant to dietary cholesterol and exhibited hepatic HMG-CoA reductase activities above 150 pmol / min(-1) / mg(-1). The Buffalo, Brown Norway, and Copenhagen 2331 rats had hepatic HMG-CoA reductase activities below 90 pmol / min(-1) / mg(-1) and had increases in serum cholesterol levels ranging from 12 to 33 mg/dl when given a 4-day, 1% cholesterol challenge. The extent of feedback regulation was reduced to only 3-fold in the Fisher 344 and Brown Norway rats that exhibited significant increases in serum cholesterol levels when given a cholesterol challenge. The Golden Syrian hamsters exhibited the largest increase (197 mg/dl) in serum cholesterol levels in response to dietary cholesterol and the lowest basal expression of hepatic HMG-CoA reductase (3.3 pmol / min(-1) / mg(-1)). Hepatic LDL receptor levels were not significantly decreased by dietary cholesterol in any of the animals. The data from these inbred rats and the hamsters strongly support the conclusion that the animals expressing the highest levels of hepatic HMG-CoA reductase are the most resistant to the serum cholesterol raising action of dietary cholesterol.     

Acyl-CoA: cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase in carp-liver microsomes: effect of cold acclimation on enzyme activities and on hepatic and plasma lipid composition.

Hepatic microsomal activities of acyl-CoA:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, rate-limiting enzymes in cholesterol esterification and cholesterol synthesis, and the concentration sand compartmentalization of esterified and unesterified cholesterol, were studied in carp acclimated to 10 and 30 degrees C. Irrespective of acclimation temperature, carp-liver ACAT is characterized by an apparent Km-value for oleoyl-CoA of 11-15 microM and displays an optimum activity at pH 7.4. The enzyme activity is reduced approx. 2-fold upon preincubation of microsomes with alkaline phosphatase. Arrhenius plots of ACAT-activity are curvilinear, with curvatures considerably affected by the acclimation temperature of the fish. Carp HMG-CoA reductase has been characterized previously by Teichert and Wodtke ((1987) Biochim. Biophys. Acta 920, 161-170). When measured at 30 degrees C, ACAT activities from 30 degrees C- and 10 degrees C-acclimated carp are identical (approx. 6 pmol/min per mg protein), whilst 'expressed' HMG-CoA reductase activity (18.1 +/- 12.2 pmol/min per mg protein for 30 degrees C-acclimated carp vs. 159.8 +/- 106.6 pmol/min per mg protein for 10 degrees C-acclimated carp) is enhanced 9-fold in the cold environment. This disparity indicates that cold-acclimation results in a massive increase in the capacity for hepatic cholesterol synthesis relative to hepatic cholesterol esterification. At the same time, hepatic compositional analysis reveals identical contents of unesterified cholesterol in either groups of carp but significantly decreased (3-fold) amounts in cholesterol ester (and also in triacylglycerol, 4-fold) in cold-acclimated carp. Moreover, microsomal fractions display lower cholesterol to phospholipid ratios in the cold. In contrast, concentrations of either cholesterol fractions (and of triacylglycerols) in plasma--the mobile compartment for lipoprotein transport--do not differ in cold- and warm-acclimated carp. Based on current concepts of cholesterol metabolism, it is concluded that the cold-enhanced expression of hepatic HMG-CoA reductase activity is a homeostatic response directed against and compensating for a cold-induced but not yet characterized deficiency in hepatic cholesterol availability.     

Cellular cholesterol metabolism in mitogen-stimulated lymphocytes--requirement for de novo synthesis

The relationship between cholesterol synthesis and uptake in proliferating lymphocytes has been examined. [14C]Acetate incorporation into lymphocytes cultured under lipoprotein-deficient conditions increased initially in response to mitogen, decreased after 24 h, and increased rapidly between 72 and 96 h. Addition of LDL (10 micrograms/ml) to the culture during the 'trough' period caused [14C]acetate incorporation to return rapidly to baseline, while at peak periods LDL suppression of cholesterol synthesis was minimal. Lymphocytes cultured in the presence of the HMG-CoA reductase inhibitor, mevinolin, exhibited a time-dependent increase in their capacity to incorporate [14C]acetate into cholesterol, evident when mevinolin was removed by washing prior to assay. PHA enhanced 125I-labelled LDL receptor-mediated binding by lymphocytes cultured in lipoprotein-deficient medium over a 4 day period and mevinolin augmented the effect. [3H]Thymidine incorporation into mitogen-stimulated lipoprotein-deficient cultures was inhibited up to 75% by mevinolin (1 mumol/l). LDL (2.5-10 micrograms/ml) substantially reversed this inhibition in 72 h cultures, but only partially overcame inhibition in cells cultured for 96 h. Results suggest that endogenous cholesterol synthesis may be obligatory for lymphocyte proliferation after the initial round of cell division.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in the human gastrointestinal tract

Activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) was measured in intestinal mucosa of the human gastrointestinal tract. Activity was highest in gastric mucosa (18.2 pmol per mg per min) and there was a constant low level in the small bowel and colon (approximately 10 pmol per mg per min). Phosphorylation/dephosphorylation modulation of intestinal reductase activity was demonstrated in normal mucosa. Expressed jejunal reductase activity was significantly higher in celiac sprue mucosa and mucosa from defunctionalized intestine of jejunoileal bypass patients. Enzyme activity also increased during 24-hr mucosal organ culture in the absence of exogenous cholesterol. Addition to the culture medium of pure cholesterol or 25-hydroxycholesterol dissolved in a small volume of ethanol suppressed the culture-induced increase to 86 +/- 3% and 69 +/- 5% of paired controls, respectively. This evidence suggests that a moderate degree of feedback regulation of intestinal cholesterol synthesis by luminal sterol occurs in man. Mucosal HMG-CoA reductase activity was also measured in patients with hyperlipoproteinemia. Patients with either predominant hypercholesterolemia or predominant hypertriglyceridemia lipid profiles had "normal" expressed reductase activity, but feedback regulation by free cholesterol could not be demonstrated in either group under these conditions.     

Coordinate regulation of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A synthase but not 3-hydroxy-3-methylglutaryl coenzyme A reductase in C-6 glia

The effects on cholesterol biosynthesis of growth of cultured C-6 glial cells in serumfree medium ± supplementation with linoleic or linolenic acid were studied. Markedly higher activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) were observed in cells grown in linoleate- or linolenate-supplemented versus nonsupplemented medium. After 48 h HMG-CoA reductase activities were two-and four-fold higher in cells supplemented with 20 and 100 μm linoleate, respectively. The increase in activity became apparent after 24 h and was marked after 48 h. Rates of incorporation of [¹⁴C]acetate or ³H₂O into sterols did not reflect the changes in reductase activity. Thus, in cells supplemented with 50 μm linoleate for 24 and 48 h rates of incorporation of [¹⁴C]acetate were 75–80% lower than rates in nonsupplemented cells. This difference resulted because over the first 24 h of the experiment a fivefold increase in the rate of sterol synthesis occurred in the nonsupplemented cells, whereas essentially no change occurred in the linoleate-supplemented cells; little further change occurred between 24 and 48 h in the nonsupplemented and the linoleate-supplemented cells. That the difference in sterol synthesis under these experimental conditions could be mediated at the level of HMG-CoA synthase (EC 4.1.3.5) was suggested by two series of findings, i.e., first, similar quantitative and temporal changes in the activity of this enzyme, and, second, no change in the activity of acetoacetyl-CoA thiolase (EC 2.3.1.9) or the incorporation of [¹⁴C]mevalonate into sterols. Thus, the data suggest that HMG-CoA synthase, and not HMG-CoA reductase, may direct the rate of cholesterol biosynthesis under these conditions of serum-free growth ± supplementation with polyunsaturated fatty acid.     

Indirect regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by oestradiol in the rabbit corpus luteum

Rabbits were given 50 i.u. hCG, i.v., to initiate ovulation and pseudopregnancy (Day 0) and were treated, s.c., with or without a 1-cm Silastic oestradiol implant. Serum progesterone concentrations were measured at 4-day intervals and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity was estimated by the conversion of HMG to mevalonate in microsomes from corpora lutea removed on Days 4, 8, 12, 16 and 20 of pseudopregnancy (4 rabbits/day). Total HMG-CoA reductase activity was significantly (P less than 0.05) higher in control rabbits on Days 8 and 12 (5.29 +/- 0.63 and 5.5 +/- 0.28 nmol/min/mg protein, respectively) compared to oestradiol-treated rabbits (2.57 +/- 0.25 and 4.03 +/- 0.23 nmol/min/mg protein, respectively). On Days 16 and 20, total HMG-CoA reductase activity was not different in control and oestradiol-treated animals. There was no difference in the levels of the active fraction of HMG-CoA reductase, which represented less than 20% of the total enzyme activity, in control and oestradiol-treated rabbits (less than 780 pmol/min/mg protein, Day 12). These results indicate that oestradiol does not alter the active form, but can reduce the total activity of HMG-CoA reductase in the rabbit corpus luteum without a decline in serum progesterone. Therefore, neither total nor active forms of HMG-CoA reductase are directly related to progesterone secretion. This suggests that other sources of cholesterol may contribute to progesterone production in the rabbit.     

The regulation of ubiquinone synthesis in fibroblasts: The effect of modulators of β-hydroxy-β-methylglutaryl-coenzyme A reductase activity

The effect of inhibitors of β-hydroxy-β-methylglutaryl-coenzyme A (HMG-CoA) reductase such as low-density lipoprotein (LDL) and compactin were tested for their effects on the biosynthesis of ubiquinone in fibroblasts using [2-¹⁴C]acetic acid as a labeled precursor. LDL added to fibroblasts incubated in lipoprotein-deficient serum inhibited acetate incorporation into ubiquinone by 35%. Compactin, 2.5 μm, inhibited acetate incorporation by 60%. Further increases in compactin concentration up to 20 μm gradually increased the extent of inhibition but leveled off between 70 and 80%. The incorporation of ³H]mevalonic acid and 4-[U-¹⁴C]hydroxybenzoic acid into ubiquinone were determined with a range of compactin concentrations. Whereas the incorporation of [³H]mevalonate showed an apparent increase in response to compactin, the incorporation of 4-[U-¹⁴C]hydroxybenzoate into ubiquinone decreased. Both curves leveled off at concentrations of 5 μm did not significantly change with further increases in compactin concentration approaching 20 μm. Thus, the inhibition of acetate and 4-hydroxybenzoate incorporation into ubiquinone by compactin showed similar patterns. Cells incubated in lipoprotein-deficient serum compared to whole human serum showed inhibition of acetate incorporation similar to that observed previously for 4-hydroxybenzoate (9), thereby suggesting the presence of a stimulatory factor for ubiquinone biosynthesis in whole human serum. These data confirm and extend our earlier conclusions that inhibition of HMG-CoA reductase greatly affects ubiquinone synthesis in fibroblasts.     

HMG-CoA reductase activity in the microsomal fraction from human placenta in early and term pregnancy

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) mevalonate: NADP oxidoreductase (CoA acylating; EC 1.1.1.34) in microsomes from early- and term-pregnancy placenta has been found to be 24 +/- 2 and 6 +/- 3 pmol/min per mg protein, respectively. Inactivation of the enzyme required the addition of ATP and Mg2+ and was dependent on the time of preincubation. Reactivation of the enzyme was also dependent on the incubation time and prevented by the presence of fluoride--a phosphoprotein phosphatase inhibitor. These data suggest that (despite a low activity) placental HMG-CoA reductase is covalently modulated via the phosphorylation-dephosphorylation system. The conversion of [14C]acetate and [3H]mevalonate into digitonin precipitable placental sterols indicates that the lower reductase activity in term, than in early, placental microsomes is accompanied by a less active conversion of [14C]acetate in this tissue.     

Green and black tea extracts inhibit HMG-CoA reductase and activate AMP kinase to decrease cholesterol synthesis in hepatoma cells

Recent studies have demonstrated that green and black tea consumption can lower serum cholesterol in animals and in man, and suppression of hepatic cholesterol synthesis is suggested to contribute to this effect. To evaluate this hypothesis, we measured cholesterol synthesis in cultured rat hepatoma cells in the presence of green and black tea extracts and selected components. Green and black tea decreased cholesterol synthesis by up to 55% and 78%, respectively, as measured by a 3-h incorporation of radiolabeled acetate. Inhibition was much less evident when radiolabeled mevalonate was used, suggesting that the inhibition was mediated largely at or above the level of HMG-CoA reductase. Both extracts directly inhibited HMG-CoA reductase when added to microsomal preparations, although the extent of inhibition was considerably less than the decrease in cholesterol synthesis observed in whole cells. As HMG-CoA reductase activity also can be decreased by enzyme phosphorylation by AMP kinase, the phosphorylation state of HMG-CoA reductase and AMP kinase, which is activated by phosphorylation, was determined in lysates from cells treated with tea extracts. Both extracts increased AMP-kinase phosphorylation and HMG-CoA reductase phosphorylation by 2.5- to 4-fold, but with different time courses: maximal phosphorylation with green tea was evident within 30 min of treatment, whereas with black tea phosphorylation was slower to develop, with maximal phosphorylation occurring > or =3 hours after treatment. These results suggest that both green and black tea decrease cholesterol synthesis in whole cells by directly inhibiting HMG-CoA reductase and by promoting its inactivation by AMP kinase.     

The regulation of hydroxymethylglutaryl-CoA reductase in cultured cells

Growth-stimulated synchronized cells exhibit a rapid increase in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.88) activity prior to the onset of DNA synthesis. Under normal culture conditions, HMG-CoA reductase activity exhibits wide variations among experiments. To determine whether this phenomenon is dependent on cell replication, we used J774 macrophage-like cells to compare changes in reductase activity in cells synchronized by serum deprivation and then growth-stimulated by fresh media containing serum to unsynchronized cells treated with fresh media and serum. Under these conditions, no increase in [3H]thymidine incorporation into cell DNA was seen in unsynchronized cells, but a large increase was observed in synchronized cells 10-12 h after media change. Although the growth characteristics differed between the cells, reductase activity was low at the time of media change and increased 10 to 20-fold 5-10 h after media change, returning to basal levels by 24 h in both synchronized and unsynchronized cells. This pattern of reductase activity was observed in unsynchronized cells from a variety of cell lineages, although the magnitude of the changes varied. Fluctuations of [14C]acetate incorporation into cholesterol were observed in parallel to alterations in reductase activity. LDL receptor expression also paralleled the changes in reductase activity, but scavenger receptor expression was not affected. Addition of lipoproteins at the time of media change inhibited the rise in reductase activity by 80-90%. The increase in reductase activity was not due to a stimulation of cholesterol efflux into the medium, but evidence for the secretion into the media of an inhibitory factor was obtained. These results suggest that cell requirements for cholesterol are not always directly related to replication, and that standard culture conditions induce transient fluctuations in reductase activity and lipoprotein receptor expression.