Tissue-selective acute effects of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase on cholesterol biosynthesis in lens

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme that regulates cholesterol synthesis, lower serum cholesterol by increasing the activity of low density lipoprotein (LDL) receptors in the liver. In rat liver slices, the dose-response curves for inhibition of [14C]acetate incorporation into cholesterol were similar for the active acid forms of lovastatin, simvastatin, and pravastatin. The calculated IC50 values were approximately 20-50 nM for all three drugs. Interest in possible extrahepatic effects of reductase inhibitors is based on recent findings that some inhibitors of HMG-CoA reductase, lovastatin and simvastatin, can cause cataracts in dogs at high doses. To evaluate the effects of these drugs on cholesterol synthesis in the lens, we developed a facile, reproducible ex vivo assay using lenses from weanling rats explanted to tissue culture medium. [14C]Acetate incorporation into cholesterol was proportional to time and to the number of lenses in the incubation and was completely eliminated by high concentrations of inhibitors of HMG-CoA reductase. At the same time, incorporation into free fatty acids was not inhibited. In marked contrast to the liver, the dose-response curve for pravastatin in lens was shifted two orders of magnitude to the right of the curves for lovastatin acid and simvastatin acid. The calculated IC50 values were 4.5 +/- 0.7 nM, 5.2 +/- 1.5 nM, and 469 +/- 42 nM for lovastatin acid, simvastatin acid, and pravastatin, respectively. Thus, while equally active in the liver, pravastatin was 100-fold less inhibitory in the lens compared to lovastatin and simvastatin. Similar selectivity was observed with rabbit lens. Following oral dosing, ex vivo inhibition of [14C]acetate incorporation into cholesterol in rat liver was similar for lovastatin and pravastatin, but cholesterol synthesis in lens was inhibited by lovastatin by as much as 70%. This inhibition was dose-dependent and no inhibition in lens was observed with pravastatin even at very high doses. This tissue-selective inhibition of sterol synthesis by pravastatin was likely due to the inability of pravastatin to enter the intact lens since pravastatin and lovastatin acid were equally effective inhibitors of HMG-CoA reductase enzyme activity in whole lens homogenates. We conclude that pravastatin is tissue-selective with respect to lens and liver in its ability to inhibit cholesterol synthesis.     

Regulation of cholesterol biosynthesis in sitosterolemia: effects of lovastatin, cholestyramine, and dietary sterol restriction.

We investigated the effects of lovastatin, cholestyramine, and dietary sterol restriction on cholesterol synthesis and low density lipoprotein receptor function in freshly isolated mononuclear leukocytes from two unrelated sitosterolemic families. Total plasma sterol concentrations were elevated in the two homozygous sitosterolemic subjects (343 and 301 vs. 185 mg/dl in controls) and contained increased amounts of plant sterols and 5 alpha-saturated stanols (20% and 8% vs. less than 1% in controls), but were not significantly different from controls in the two heterozygous subjects. The rates of conversion of acetate to cholesterol by mononuclear leukocytes were subnormal in all homozygous and heterozygous subjects and correlated with markedly reduced microsomal 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase activity. In the two homozygous subjects, cholestyramine treatment decreased plasma sterols 29% and 35%, and yet was associated with a paradoxical decline in mononuclear leukocyte HMG-CoA reductase activity. In contrast, plasma sterol concentrations decreased 14% and 5%, and mononuclear leukocyte HMG-CoA reductase activities increased 13% and 46% in three control and one heterozygous subjects treated with cholestyramine, respectively. Plasma sterol concentrations in the homozygous subjects unexpectedly failed to decline during treatment with lovastatin or a low sterol diet. In distinction, plasma sterol concentrations in three control and one heterozygous subjects dropped 28% and 31%, respectively, during treatment with lovastatin. Both cholestyramine and low dietary sterols stimulated low density lipoprotein receptor function. These results demonstrate a marked abnormality in cholesterol homeostasis in patients with homozygous sitosterolemia with xanthomatosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Effects of lovastatin on biliary lipid secretion and bile acid metabolism in humans

Lovastatin, an inhibitor of HMG-CoA reductase, lowers cholesterol saturation of bile. To determine the mechanism of this effect and further define the role of cholesterol synthesis in regulation of biliary lipid metabolism, we studied ten human volunteers in a control period and again after 5-6 weeks on lovastatin, 40 mg b.i.d. Mean sterol production from acetate in mononuclear leukocytes fell from 1.18 to 0.84 pmol/min per 10(6) cells on lovastatin (P less than 0.02). Concomitantly there was reduction in mean biliary secretion of cholesterol from 143 to 96 mumol/h (P less than 0.02). On lovastatin, mean pool size of bile acids by the Lindstedt method fell from 3193 to 2917 mumol (one-sided P = 0.05) and mean pool size by the one-sample method fell from 5158 to 4091 mumol (P less than 0.002). Lovastatin had no effect on mean fractional turnover rate of either cholic acid (0.77 vs. 0.74 day-1) or chenodeoxycholic acid (0.51 vs. 0.54 day-1). Mean total bile acid synthesis was lower on lovastatin (1443 vs. 1240 mumol/day), but the difference did not quite achieve statistical significance. In humans, inhibition of cholesterol synthesis by lovastatin lowers biliary cholesterol saturation by reducing cholesterol secretion into bile. Bile acid pool size, and perhaps bile acid synthesis, are also reduced by this inhibition.     

Hypercholesterolaemia: simvastatin and pravastatin alter cholesterol metabolism by different mechanisms

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor simvastatin, reduced low-density-lipoprotein (LDL) cholesterol in hypercholesterolaemic patients by 40% (P less than 0.001). The reduction in LDL cholesterol was accompanied by a significant decrease in the esterified/free cholesterol ratio of the patients' LDL from 2.51 +/- 0.13 to 2.06 +/- 0.14 (P less than 0.01). This change led to a significant increase (P less than 0.05) in the capacity of the LDL to suppress [14C]acetate incorporation into cholesterol in mononuclear leucocytes. Furthermore, [14C]acetate incorporation into the patients mononuclear leucocytes was significantly lower (P less than 0.02) following drug treatment (117 +/- 22 vs. 162 +/- 29 nmol/mg cell protein). Comparison of simvastatin with another HMG-CoA reductase inhibitor pravastatin, showed similar reduction in LDL cholesterol. Pravastatin treatment however, did not result in a reduction in the LDL esterified/free cholesterol ratio or in the changes in cellular cholesterol synthesis and its regulation by LDL which accompanied simvastatin treatment. The activity of the enzyme acyl-coenzyme A: cholesterol acyltransferase (ACAT) in patients' mononuclear cells remained unchanged after treatment with either drug. Results of the study show that while the drugs are equally effective in lowering LDL cholesterol, simvastatin has additional compositional effects on LDL which increase its capacity to regulate mononuclear leucocyte cholesterologenesis.     

Role of cholesterol synthesis in regulation of bile acid synthesis and biliary cholesterol secretion in humans

We used lovastatin, a specific inhibitor of HMG-CoA reductase, to study the role of cholesterol synthesis in regulation of both bile acid synthesis, measured by release of 14CO2 from [26-14C]cholesterol, and biliary cholesterol secretion, measured by standard marked perfusion techniques, in humans. Six volunteers were studied in each of four periods: a) control; b) 6-10 hours after a single 40 mg oral dose of lovastatin to study acute effects; c) after 5-6 weeks of lovastatin 40 mg orally twice a day to study steady-state effects; and d) 24 h after cessation of chronic lovastatin. Mean bile acid synthesis fell to 69% of control (P less than 0.01) after single-dose lovastatin and remained at 83% of control after 5-6 weeks on lovastatin (P less than 0.05). After withdrawal of lovastatin, mean bile acid synthesis was 88% of control (NS). Mean biliary cholesterol secretion did not change after single-dose lovastatin (103% of control), but fell to 81% of control during chronic lovastatin treatment (P less than 0.05). After withdrawal of lovastatin, mean cholesterol secretion remained at 80% of control (P less than 0.05). These data suggest that in humans cholesterol synthesis is an immediate regulator of bile acid synthesis. Cholesterol synthesis also regulates biliary cholesterol secretion, but the effect is not immediate and therefore may be indirect.     

Hepatic and nonhepatic sterol synthesis and tissue distribution following administration of a liver selective HMG-CoA reductase inhibitor, CI-981: comparison with selected HMG-CoA reductase inhibitors.

Since cholesterol biosynthesis is an integral part of cellular metabolism, several HMG-CoA reductase inhibitors were systematically analyzed in in vitro, ex vivo and in vivo sterol synthesis assays using [14C]acetate incorporation into digitonin precipitable sterols as a marker of cholesterol synthesis. Tissue distribution of radiolabeled CI-981 and lovastatin was also performed. In vitro, CI-981 and PD134967-15 were equipotent in liver, spleen, testis and adrenal, lovastatin was more potent in extrahepatic tissues than liver and BMY21950, pravastatin and PD135023-15 were more potent in liver than peripheral tissues. In ex vivo assays, all inhibitors except lovastatin preferentially inhibited liver sterol synthesis; however, pravastatin and BMY22089 were strikingly less potent in the liver. CI-981 inhibited sterol synthesis in vivo in the liver, spleen and adrenal while not affecting the testis, kidney, muscle and brain. Lovastatin inhibited sterol synthesis to a greater extent than CI-981 in the spleen, adrenal and kidney while pravastatin and BMY22089 primarily affected liver and kidney. The tissue distribution of radiolabeled CI-981 and lovastatin support the changes observed in tissue sterol synthesis. Thus, we conclude that a spectrum of liver selective HMG-CoA reductase inhibitors exist and that categorizing agents as liver selective is highly dependent upon method of analysis.     

Selective inhibition of cholesterol synthesis in liver versus extrahepatic tissues by HMG-CoA reductase inhibitors

Hepatic specificity of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase may be achieved by efficient first-pass liver extraction resulting in low circulating drug levels, as with lovastatin, or by lower cellular uptake in peripheral tissues, seen with pravastatin. BMY-21950 and its lactone form BMY-22089, new synthetic inhibitors of HMG-CoA reductase, were compared with the major reference agent lovastatin and with the synthetic inhibitor fluindostatin in several in vitro and in vivo models of potency and tissue selectivity. The kinetic mechanism and the potency of BMY-21950 as a competitive inhibitor of isolated HMG-CoA reductase were comparable to the reference agents. The inhibitory potency (cholesterol synthesis assayed by 3H2O or [14C]acetate incorporation) of BMY-21950 in rat hepatocytes (IC50 = 21 nM) and dog liver slices (IC50 = 23 nM) equalled or exceeded the potencies of the reference agents. Hepatic cholesterol synthesis in vivo in rats was effectively inhibited by BMY-21950 and its lactone form BMY-22089 (ED50 = 0.1 mg/kg p.o.), but oral doses (20 mg/kg) that suppressed liver synthesis by 83-95% inhibited sterol synthesis by only 17-24% in the ileum. In contrast, equivalent doses of lovastatin markedly inhibited cholesterol synthesis in both organs. In tissue slices from rat ileum, cell dispersions from testes, adrenal, and spleen, and in bovine ocular lens epithelial cells, BMY-21950 inhibited sterol synthesis weakly in vitro with IC50 values 76- and 188-times higher than in hepatocytes; similar effects were seen for BMY-22089. However, the IC50 ratios (tissue/hepatocyte) for lovastatin and fluindostatin were near unity in these models. Thus, BMY-21950 and BMY-22089 are the first potent synthetic HMG-CoA reductase inhibitors that possess a very high degree of liver selectivity based upon differential inhibition sensitivities in tissues. This cellular uptake-based property of hepatic specificity of BMY-21950 and BMY-22089, also manifest in pravastatin, is biochemically distinct from the pharmacodynamic-based disposition of lovastatin, which along with fluindostatin exhibited potent inhibition in all tissues that were exposed to it.     

Naringin alters the cholesterol biosynthesis and antioxidant enzyme activities in LDL receptor-knockout mice under cholesterol fed condition

The purpose of the current study was to evaluate the lipid lowering and antioxidant capacity of naringin in LDL receptor knockout (LDLR-KO) mice fed a cholesterol (0.1 g/100 g) diet. As such, naringin or lovastatin (0.02 g/100 g) was supplemented in a cholesterol diet for 6 weeks. The naringin and lovastatin supplementation significantly lowered the plasma total cholesterol level compared to the control group. The plasma and hepatic triglyceride level was only lowered by the lovastatin supplement, while the hepatic cholesterol content was lowered by both the naringin and lovastatin supplements compared to the control group. The hepatic HMG-CoA reductase activity was significantly lower in the naringin and lovastatin supplemented groups than in the control group, whereas the ACAT activity was unaffected. The excretion of total sterol was significantly higher in the naringin and lovastatin groups compared to the control group due to significant changes in the acidic and neutral sterol, respectively. When comparing the hepatic antioxidant enzyme activities, the superoxide dismutase, catalase, and glutathione reductase activities were all significantly higher in the naringin-supplemented group than in the control group, while only the lovastatin supplement increased the glutathione reductase activity. Accordingly, the current results confirmed that naringin lowers the plasma cholesterol level via the inhibition of hepatic HMG-CoA reductase activity and increases the excretion of fecal sterol. Naringin was also found to improve the activities of hepatic antioxidant enzymes against oxidative stress in a hypercholesterolemic animal model, i.e. cholesterol-fed LDLR-KO mice.     

Regulation of bile acid synthesis. IV. Interrelationship between cholesterol and bile acid biosynthesis pathways

Under most experimental conditions, the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase) and cholesterol 7 alpha-hydroxylase, change together in parallel directions. It has been suggested that newly synthesized cholesterol may be the preferred substrate for cholesterol 7 alpha-hydroxylase, which may account for the observed synchronous behavior of the two enzymes. To test this hypothesis, mevinolinic acid, a potent competitive inhibitor of HMG-CoA reductase, was administered as a single intravenous bolus (10 mg/kg) to rats with a chronic bile fistula. Bile acid synthesis was determined following inhibition of HMG-CoA reductase by mevinolinic acid over a 27-h time course and specific activities of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase were determined in liver microsomes. At 3, 6, and 27 h after a bolus dose of mevinolinic acid, bile acid synthesis was reduced by 54 +/- 5%, 42 +/- 8%, and 23 +/- 13%, respectively, from preinfusion baseline. Within 30 min after administration of mevinolinic acid, HMG-CoA reductase activity was inhibited by at least 87%. At 0.5, 1.5, 3, 6, and 27 h after mevinolinic acid injection, cholesterol 7 alpha-hydroxylase activity was decreased by 6%, 25%, 54%, 41%, and 17%, respectively. By 27 h, the activities of both enzymes had returned to baseline levels. The reduction of bile acid synthesis correlated closely with the observed changes in the activities of cholesterol 7 alpha-hydroxylase. In vitro addition of mevinolinic acid (up to 20 microM) to rat liver microsomes failed to inhibit cholesterol 7 alpha-hydroxylase activity, suggesting no direct effect of mevinolinic acid on enzyme activity. When a bolus dose of mevinolinic acid was coupled with a continuous infusion of mevalonate, the product of the reaction catalyzed by HMG-CoA reductase, the mevinolinic acid-induced decrease in cholesterol 7 alpha-hydroxylase activity and bile acid synthesis was prevented. The results of this study provide evidence that, under the experimental conditions described, there is a linkage between the rates of cholesterol synthesis and the activities of cholesterol 7 alpha-hydroxylase. The data also emphasize the importance of the newly synthesized cholesterol in the regulation of cholesterol 7 alpha-hydroxylase activity.     

Variations of hepatic cholesterol precursors during altered flows of endogenous and exogenous squalene in the rat

Hepatic and serum levels of cholesterol precursors were analyzed in rats under basal (control) conditions and when cholesterol synthesis was activated by feeding 1% squalene or 5% cholestyramine. Exogenous squalene stimulated the activity of acyl-coenzyme A:cholesterol acyltransferase (ACAT) but strongly inhibited the activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase; cholestyramine did not affect ACAT but increased HMG-CoA reductase several-fold, indicating enhanced production of endogenous squalene. Activation of cholesterol synthesis by the two methods markedly increased the hepatic and serum contents of cholesterol precursor sterols. However, the sterol profiles were clearly different. Thus, exogenous squalene raised most significantly (up to 109-fold) free and esterified methyl sterols, and less so (up to 2-fold) demethylated C27 sterols (desmosterol and cholestenols) and also esterified cholesterol. Activation of endogenous squalene production by cholestyramine was associated with a depletion of esterified cholesterol and by a marked, up to 8-fold, increase of the free demethylated sterol precursor levels, whereas the increase of methyl sterols, up to 5-fold, was less conspicuous than during the squalene feeding. The changes were mostly insignificant for esterified sterols. The altered serum sterol profiles were quite similar to those in liver. Serum cholestenols and especially their portion of total serum precursor sterols were closely correlated with the hepatic activity of HMG-CoA reductase.     

Whole body and hepatic cholesterol synthesis rates in the guinea-pig: effect of dietary fat quality

The effects of polyunsaturated, monounsaturated and saturated dietary fat on total and hepatic cholesterol synthesis were studied in the guinea-pig. Male Hartley guinea-pigs were fed semi-synthetic diets containing 7.5% (w/w) of either corn oil (CO), olive oil (OL) or lard for a period of 5 weeks and rates of endogenous cholesterol synthesis were determined from the incorporation of [3H]water into digitonin-precipitable sterols (DPS) and by measurement of sterol balance. In addition, total and expressed 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities were determined in hepatic microsomes. Rates of whole body cholesterol synthesis determined by incorporation of [3H]water into DPS were significantly lower for guinea-pigs on the CO diet with values of 18.7 +/- 1.8 mumol/h (n = 4) vs. 26.7 +/- 4.8 and 24.6 +/- 1.8 mumol/h for animals on the OL (n = 4) and lard (n = 3) diets (P less than 0.001), respectively. Hepatic cholesterol synthesis rates were significantly decreased in animals on the OL diet, whether determined from incorporation of [3H]water into DPS or by analysis of HMG-CoA reductase activity. Hepatic total and free cholesterol levels were not different for animals on the three dietary fats; however, cholesteryl ester levels were 35% lower in guinea-pigs fed the lard diet (P less than 0.02). Sterol balance measurements indicated that whole body cholesterol synthesis rates were not affected by dietary fat quality (51.9 +/- 12.2, 42.8 +/- 7.6 and 51.2 +/- 20.2 mg/kg per day for animals on the CO, OL and lard diets, respectively). This is in striking contrast to the observed reduction in cholesterol synthesis rates for animals on the polyunsaturated CO diet as determined by incorporation of [3H]water into DPS. One possible explanation for the discrepancy between the sterol balance and [3H]water incorporation data is a polyunsaturated fat-mediated effect on energy utilization, which affects the equilibration of NADPH with the body water pool such that the [3H]NADPH has a lower specific activity than body [3H]water.     

Mechanisms for cholesterol homeostasis in rat jejunal mucosa: effects of cholesterol, sitosterol, and lovastatin

The effects of feeding cholesterol, sitosterol, and lovastatin on cholesterol absorption, biosynthesis, esterification, and LDL receptor function were examined in the rat jejunal mucosa. Cholesterol absorption was measured by the dual-isotope plasma ratio method; the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, was measured as total and expressed enzyme activities (in the absence and presence of a phosphatase inhibitor, NaF, respectively); mucosal total and esterified cholesterol concentrations were determined by gas-liquid chromatography; LDL receptor function was assayed as receptor-mediated binding of (125)I-labeled LDL to mucosal membranes. Feeding 2% sitosterol or 0.04% lovastatin for 1 week significantly (P < 0.01) decreased the amounts of cholesterol absorbed per day (-85% and -63%, respectively). In contrast, feeding 2% cholesterol for 1 week increased the amounts of absorbed cholesterol 27-fold, even though the percent absorption significantly decreased. With all three treatments, there was a coordinate regulation of total HMG-CoA reductase activity and receptor-mediated LDL binding. Cholesterol feeding downregulated both total jejunal HMG-CoA reductase activity (P < 0.05) and receptor-mediated LDL binding (P < 0.01), whereas lovastatin- and sitosterol-supplemented diets significantly upregulated both of these parameters. In the control, cholesterol-fed, and sitosterol-fed animals, about half of the total jejunal HMG-CoA reductase activity was expressed (in functional dephosphorylated form). However, in the lovastatin-treated rats with 4-fold stimulation of HMG-CoA reductase, only 23% of the total enzyme activity was expressed. Changes in total HMG-CoA reductase activity and receptor-mediated LDL binding in all tested groups occurred with no change in total concentrations of mucosal cholesterol, and only cholesterol-fed animals had increased mucosal esterified cholesterol concentrations. Thus, in response to various fluxes of dietary or newly formed cholesterol, HMG-CoA reductase and receptor-mediated LDL binding are coordinately regulated to maintain constant cellular cholesterol concentrations in the jejunum.     

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in hepatoma tissue culture cells by pure cholesterol and several cholesterol derivatives. Evidence supporting two distinct mechanisms.20l.

Pure cholesterol associated in complexes with lipoproteins (whole serum and human low density lipoproteins) or esterified with succinic acid (cholesteryl succinate) and bound to albumin effectively suppresses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in hepatoma tissue culture (HTC) cells grown in lipoprotein-poor serum medium during short 4-hour) incubation periods. Simultaneous measurments of the kinetics of uptake of radioactive unesterified cholesterol of whole serum and cholesteryl succinate, their conversion to lipid products, and the decay in enzyme activity, suggest that the cholesterol-induced suppression is mediated by the sterol itself rather than by inhibitory lipid products derived from its metabolism. Several cholesterol derivatives such as cholestenone, 7-ketocholesterol, and 7alpha-and 25-hydroxycholesterol also suppress reductase activiy in HTC cells and are significantly more inhibitory than the pure cholesterol preparations. The decrease in enzyme activity produced by cholesterol and its derivatives is concentration-dependent and specific. [1-14C]Oleate incorporation experiments indicate that cholesterol ester formation in HTC cells is not increased at inhibitory concentrations of the steroids. These data suggest that sterol ester formation is not an obligatory process in the feedback control of HMG-CoA reductase activity. The half-life of the reductase (3 to 4 hours) is not significantly changed by cycloheximide, plus or minus whole serum, and cholesteryl succinate. In contrast, the half-life is strongly reduced when HTC cells are incubated with cycloheximide plus maximal concentrations of 25-hydroxycholesterol, 7-ketocholesterol, or cholestenone, resulting in t1/2 values of 24, 36, and 60 min, respectively. Increasing concentrations of whole serum and cholesteryl succinate have no significant effect on the apparent rate constant of inactivation of the enzyme, whereas its apparent rate of synthesis is decreased 3- and 10-fold, respectively. These results are reversed with oxygenated steroid inhibitors. The rate of synthesis of reductase is essentially unchanged as the concentrations of 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone are increased in the culture medium, whereas the apparent rate constant for degradation is increased 9-, 7-, and 3-fold, respectively. HMG-CoA reductase activity in HTC cells thus appears to be modulated by two different mechanisms in which steroid structure is important. Whole serum and cholesteryl succinate specifically decrease the rate of enzyme synthesis, while 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone increase the rate of inactivation of the reductase.     

Effect of lovastatin on acyl-CoA: cholesterol O-acyltransferase (ACAT) activity and the basolateral-membrane secretion of newly synthesized lipids by CaCo-2 cells.

Lovastatin, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity, was used to study the regulation of cholesterol metabolism and the basolateral-membrane secretion of triacylglycerol and cholesterol in the human intestinal cell line CaCo-2. At 0.1 microgram/ml, lovastatin decreased 3H2O incorporation into cholesterol by 71%. In membranes prepared from cells incubated with lovastatin for 18 h, HMG-CoA reductase activity was induced 4-8-fold. Mevalonolactone prevented this induction. In intact cells, lovastatin (10 micrograms/ml) decreased cholesterol esterification by 50%. The reductase inhibitor decreased membrane acyl-CoA:cholesterol O-acyltransferase (ACAT) activity by 50% at 5 micrograms/ml. ACAT inhibition by lavastatin was not reversed by adding excess of cholesterol or fatty acyl-CoA to the assay. Lovastatin, in the presence or absence of mevalonolactone, decreased the basolateral secretion of newly synthesized cholesteryl esters and triacylglycerols. Lovastatin also inhibited the esterification of absorbed cholesterol and the secretion of this newly synthesized cholesteryl ester. Lovastatin is a potent inhibitor of cholesterol synthesis in CaCo-2 cells. Moreover, it is a direct inhibitor of ACAT activity, independently of its effect on HMG-CoA reductase and cholesterol synthesis.     

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.     

Regulation of cholesterol synthesis in cultured mouse mammary carcinoma FM3A cells

Mouse mammary carcinoma FM3A cells, which are able to grow in a serum-free medium, have novel characteristics that could be valuable in biochemical and somatic cell genetic studies. In FM3A cells grown in the presence of serum, both sterol synthesis and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the major rate-limiting enzyme in the cholesterol biosynthetic pathway, were strongly suppressed by human low density lipoprotein (LDL). The addition of LDL (50 micrograms protein/ml) resulted in a 50% decrease in the reductase activity within 3 h and a 95% reduction after 24 h. Similarly, over 90% suppression of the reductase activity was obtained by the addition of LDL or mevalonolactone when the cells were grown on a serum-free medium. ML-236B (compactin), a specific inhibitor of HMG-CoA reductase, inhibited sterol synthesis from [14C]acetate by 80% at 1 microM. Reductase activity in FM3A cells was increased by 2.5- to 5-fold when the cells were treated with ML-236B (at 0.26-2.6 microM for 24 h). Thus, in FM3A cells, HMG-CoA reductase activity responded well to LDL, as is observed in human skin fibroblasts. Along with other novel features of this cell line, the present observations indicate that FM3A cells should be useful in biochemical and somatic cell genetic analysis of cholesterol metabolism, especially as regards the regulation of HMG-CoA reductase activity.     

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.