Effect of ACTH on cholesterol and steroid synthesis in adrenocortical tissues

Previous studies have established that under normal conditions, adrenal HMG-CoA reductase activity is higher in hamsters than in rats and humans. The hamster reductase activity follows a diurnal rhythm corresponding to that of plasma ACTH and glucocorticoids [Endocrinology 107 (1980) 215] but not to that of aldosterone. ACTH treatments to hamsters increased reductase activity after a latency of 60 min; this enhancement was prevented by cycloheximide [J. steroid Biochem. 24 (1986) 325]. Immunotitration and immunoblotting studies confirmed that ACTH caused an increase in reductase protein synthesis. In rats, long-term (1-9 days) and short-term (3 h) treatments with ACTH also induced increase in adrenal HMG-CoA reductase activity and reductase protein. In the presence of iodoacetamide and inhibitors of proteolytic enzyme, a main specific band of enzyme was evinced in the area of 102 +/- 6 kDaMr, by Western blotting, for both hamster homogenate and microsomal preparations (Endocrinology, 120 (1987]. Similarly Mr values were found with rat adrenal preparations. The concentration of mRNA, analyzed using the c-DNA pRed-10 coding for the Chinese hamster ovary reductase, was increased in adrenals of hamsters treated with ACTH. The reductase mRNA levels also fluctuated during the day in parallel with those of reductase activity and reductase protein. In conclusion, these results indicate that ACTH and other conditions inducing a change in hamster adrenal HMG-CoA reductase activity provoke parallel changes in reductase mRNA and reductase protein content. ACTH acts on the adrenal reductase of species synthesizing large as well as small quantities of cholesterol, thus indicating the general importance of this hormonal control.     

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.     

3-hydroxy-3-methylglutaryl coenzyme A reductase in human liver microsomes: active and inactive forms and cross-reactivity with antibody against rat liver enzyme

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the enzyme catalyzing the rate-limiting step in cholesterol biosynthesis, exists in one active (dephosphorylated) and one inactive (phosphorylated) form in liver microsomes obtained from several animal species. The present study was undertaken in order to determine a) whether the human enzyme also exists in active and inactive readily interconvertible forms; b) whether the large inter-individual variation in HMG-CoA reductase activity observed in normal man can be explained by variations in the activation state of the enzyme; and c) to characterize the reactivity of antibodies raised against rat liver HMG-CoA reductase with the intact human microsomal enzyme. HMG-CoA reductase activity, assayed in microsomes prepared in the presence of 50 mM NaF, was only 17 +/- 3% of the activity observed in microsomes prepared from the same liver in the absence of fluoride. Preincubation of microsomes prepared in NaF with alkaline phosphatase resulted in a tenfold increase of enzyme activity, while the activity of microsomes prepared without fluoride was increased also (by about 45%) with this treatment. On the other hand, the activated enzyme could be inactivated by incubation of microsomes with Mg-ATP. In eleven normal weight, normolipidemic gallstone patients, the HMG-CoA reductase activity determined in microsomes prepared without NaF ("standard procedure") reflected well both the "expressed" activity (in microsomes prepared with NaF) and the "total" (fully activated) enzyme activity; correlation coefficients were +0.80 and +0.84, respectively. Preincubation of human liver microsomes with rabbit antiserum against partially purified HMG-CoA reductase from rat liver resulted in a 72 +/- 6% inhibition of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of 3-hydroxy-3-methylglutaryl coenyzyme a (HMG-CoA) reductase activity with intracellular fractions of hamster adrenals

Hamster adrenal HMG-CoA reductase activity was enhanced with rat liver cytosolic phosphorylase phosphatase as well as with similarly isolated beef and hamster adrenal cytosolic preparations. HMG-CoA reductase was inactivated when microsomes were incubated in an EDTA-free medium but containing MgCl₂ and ATP. The reductase activity of microsomes isolated from adrenals of hamsters sacrificed at 1100 h and 1900 h were (mean ± SEM, pmo1/mg protein/min.) 299.6±62.3 and 588.3 ± 96.6 respectively and could be enhanced by a factor of four when preincubated in the presence of liver phosphatase.     

Lovastatin treatment inhibits sterol synthesis and induces HMG-CoA reductase activity in mononuclear leukocytes of normal subjects

The mechanism by which competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase decrease serum cholesterol is incompletely understood. The few available data in humans suggest that chronic administration of the competitive inhibitor, lovastatin, decreases serum cholesterol with little or no change in total body sterol synthesis. To further define the effect of lovastatin on cholesterol synthesis in normal subjects, we investigated the effect of a single oral dose of lovastatin and a 4-week treatment period of lovastatin on mononuclear leukocyte (ML) sterol synthesis as a reflection of total body sterol synthesis. In parallel, we measured serum lipid profiles and HMG-CoA reductase activity in ML microsomes that had been washed free of lovastatin. ML sterol synthesis did not significantly decrease (23 +/- 5%, mean +/- SEM) at 3 h after a single 40-mg dose of lovastatin. With a single oral 80-mg dose, ML sterol synthesis decreased by 57 +/- 10% (P less than 0.05) and remained low for the subsequent 6 h. With both doses, total HMG-CoA reductase enzyme activity in microsomes prepared from harvested mononuclear leukocytes was induced 4.8-fold (P less than 0.01) over baseline values. Both the 20-mg bid dose and the 40-mg bid dose of lovastatin administered for a 4-week period decreased serum cholesterol by 25-34%. Lovastatin at 20 mg bid decreased ML sterol synthesis by 23 +/- 6% (P less than 0.02) and increased ML HMG-CoA reductase 3.8 times (P less than 0.001) the baseline values. Twenty four hours after stopping lovastatin, ML sterol synthesis and HMG-CoA reductase enzyme activity had returned to the baseline values. The higher dose of lovastatin (40 mg bid) decreased ML sterol synthesis by 16 +/- 3% (P less than 0.05) and induced HMG-CoA reductase to 53.7 times (P less than 0.01) the baseline value at 4 weeks. Stopping this higher dose effected a rebound in ML sterol synthesis to 140 +/- 11% of baseline (P less than 0.01), while HMG-CoA reductase remained 12.5 times baseline (P less than 0.01) over the next 3 days. No rebound in serum cholesterol was observed. From these data we conclude that in normal subjects lovastatin lowers serum cholesterol with only a modest effect on sterol synthesis. The effect of lovastatin on sterol synthesis in mononuclear leukocytes is tempered by an induction of HMG-CoA reductase enzyme quantity, balancing the enzyme inhibition by lovastatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hormonal regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA in the rat adrenal gland

We studied the effect of ACTH on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase enzyme. Reductase activity and reductase mass were enhanced by 22- and 6.2-fold respectively in one series of experiments, whereas in another the levels of reductase activity, reductase mass, and reductase mRNA were increased 6.6-, 3.6- and 2.2-fold respectively, following daily administration of exogenous ACTH for 3 days. Daily injection of 4-aminopyrazolopyrimidine (4-APP) to rats for 3 days increased circulating ACTH level 5.4-fold, whereas adrenal HMG-CoA reductase activity, reductase mass and reductase mRNA levels were greatly increased 36-, 10- and 16-fold, respectively. To counteract the effect of elevated plasma ACTH, dexamethasone acetate (Dex) was administered to 4-APP treated rats. At 3 h post Dex administration, plasma ACTH and corticosteroids levels were effectively decreased by 58 and 59%, respectively. The levels of adrenal HMG-CoA reductase mRNA, reductase activity and reductase mass were also diminished by 38, 31 and 40%, respectively. Our results show that rat adrenal HMG-CoA reductase can respond rapidly to hormonal changes, presumably through variations in circulating ACTH levels.     

Properties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and regulation of enzyme activity

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from rat liver microsomes has been purified to apparent homogeneity with recoveries of approximately 50%. The enzyme obtained from rats fed a diet supplemented with cholestyramine had specific activities of approximately 21,500 nmol of NADPH oxidized/min/mg of protein. After amino acid analysis a specific activity of 31,000 nmol of NADPH oxidized/min/mg of amino acyl mass was obtained. The s20,w for HMG-CoA reductase was 6.14 S and the Stokes radius was .39 nm. The molecular weight of the enzyme was 104,000 and the enzyme subunit after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 52,000. Antibodies prepared against the homogeneous enzyme specifically precipitated HMG-CoA reductase from crude and pure fractions of the enzyme. Incubation of rat hepatocytes for 3 h in the presence of lecithin dispersions, compactin, or rat serum resulted in significant increases in the specific activity of the microsomal bound reductase. Immunotitrations indicated that in all cases these increases were associated with an activated form of the reductase. However activation of the enzyme accounted for only a small percentage of the total increase in enzyme activity; the vast majority of the increase was apparently due to an increase in the number of enzyme molecules. In contrast, when hepatocytes were incubated with mevalonolactone the lower enzyme activity which resulted was primarily due to inactivation of the enzyme with little change in the number of enzyme molecules. Immunotitrations of microsomes obtained from rats killed at the nadir or peak of the diurnal rhythm of 3-hydroxy-3-methylglutaryl-CoA reductase indicated that the rhythm results both from enzyme activation and an increased number of reductase molecules.     

ACTH induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase, cholesterol biosynthesis, and steroidogenesis in primary cultures of bovine adrenocortical cells

The current studies demonstrate that corticosteroidogenesis can be maintained by primary cultures of bovine adrenocortical cells under lipoprotein-depleted conditions. The cholesterol necessary as substrate for steroid synthesis was found to arise from de novo synthesis within these cells. Adrenocorticotropin (ACTH) increased 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity 5-fold within 12 h after addition to the medium. The increase in activity apparently represented accumulation of enzyme as determined by protein blotting and immunodetection. The predominant immunodetectable species of HMG-CoA reductase from bovine adrenal cells was 97,000 daltons; no higher molecular mass species was detectable. The ACTH induction of HMG-CoA reductase activity could be prevented after inhibition of cholesterol conversion to pregnenolone with clotrimazole. These results are suggestive that ACTH increases adrenocortical cholesterol biosynthesis and HMG-CoA reductase activity after conversion of a cellular pool of cholesterol and/or oxysterol into steroid. The increased rate of cholesterol biosynthesis is then capable of maintaining ACTH-promoted steroid production. This is the first study, in vitro, to demonstrate an ACTH-promoted accumulation of HMG-CoA reductase of adrenocortical cells.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in avian myeloblasts. Mode of action of 25-hydroxycholesterol

25-Hydroxycholesterol inhibits cholesterol biosynthesis by inhibiting the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Addition of 25-hydroxycholesterol to chicken myeloblasts caused a rapid inhibition of HMG-CoA reductase activity, producing approximately an 80% decrease in enzyme activity after 60 min. The mode of action of 25-hydroxycholesterol was determined by immunoprecipitating radiolabeled enzyme from 25-hydroxycholesterol-treated myeloblasts. The decline in enzyme activity due to addition of 25-hydroxycholesterol was not associated with increased levels of [32P]PO4 incorporation into the immunoprecipitated reductase polypeptide (Mr = 94,000). Hence, 25-hydroxycholesterol did not appear to regulate reductase activity by enzyme phosphorylation, as observed for other modulators of HMG-CoA reductase. However, 25-hydroxycholesterol was shown to inhibit reductase activity by causing a 350% increase in the relative rate of reductase degradation and a 72% decrease in the relative rate of reductase synthesis. These alterations in the rates of degradation and synthesis occurred rapidly (within 10-30 min after addition of 25-hydroxycholesterol) and can account completely for the 25-hydroxycholesterol-induced inhibition of enzyme activity. The rapid decline in the rate of synthesis of HMG-CoA reductase in 25-hydroxycholesterol-treated cells was not associated with concomitant changes in the levels of reductase mRNA; therefore, suggesting that 25-hydroxycholesterol must inhibit the rate of reductase synthesis by translational regulation. We also present evidence that mRNA purified from chicken myeloblasts codes for two reductase polypeptides of Mr = 94,000 and 102,000.     

Regulation of 3-hydroxyl-3-methylglutaryl-coenzyme A reductase activity in rat uterine tissues

Initiation of uterine DNA synthesis and mitosis in response to estrogen appears to depend upon the stimulation of protein synthesis. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase could have a key function in controlling uterine mitosis through its control of mevalonic acid and cholesterol synthesis as the rate-limiting enzyme in their synthetic pathways. These studies were initiated to examine the kinetics of the uterine increases in HMG-CoA reductase activity in response to estradiol. In the uterus of the ovariectomized mature rat, estradiol increased levels of enzyme activity in both the luminal epithelium and stroma-myometrium up to 12 h after estradiol treatment. Levels of HMG-CoA reductase activity decreased after 12 h in the luminal epithelium and further increased in the stroma-myometrium. Previous studies have shown that estradiol does not increase DNA synthesis and mitosis in the stroma-myometrium of the uterus of the ovariectomized mature rat. Since estradiol increased HMG-CoA reductase activity in both the luminal epithelium and stroma-myometrium, we conclude that even though increased HMG-CoA reductase activity may be a prerequisite for increased DNA synthesis, increases in uterine HMG-CoA reductase activity are not necessarily followed by increased DNA synthesis.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase in the sea urchin embryo is developmentally regulated

The activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, an enzyme which plays a regulatory role in the synthesis of cholesterol, dolichol, and coenzyme Q, has been measured in the developing embryo of the sea urchin. Enzyme activity increased at least 200-fold during development from the unfertilized egg to the pluteus stage embryo. Mixing experiments suggested that the low level of enzyme activity found at early stages was not due to the presence of inhibitor(s) in the egg or zygote. The enzyme in the sea urchin embryo exhibited properties different from that found in mammals: only a fraction of the activity could be solubilized from microsomes, and mild trypsinization inactivated the enzyme without releasing any of it from the microsomes in soluble form. To further study the sea urchin HMG-CoA reductase, a genomic clone was identified by hybridization to a cDNA encoding hamster HMG-CoA reductase. Sequence analysis of this clone revealed a coding region that shares a high degree of homology with the carboxyl-terminal domain of hamster HMG-CoA reductase. Analysis of sea urchin embryo HMG-CoA reductase mRNA levels using a restriction fragment derived from the genomic clone revealed a 5.5-kilobase poly(A)+ mRNA that increased 15-fold during development from the egg to the gastrula stage and then decreased 1.5-fold at the pluteus stage. Since the relative increase in HMG-CoA reductase mRNA was less than the increase in enzyme activity (15-fold versus 200-fold) factors in addition to the level of mRNA may control the activity of this enzyme during embryogenesis.     

Rapid modulation of rat hepatocyte HMG-CoA reductase activity by cyclic AMP or cyclic GMP

Rat hepatocytes were used to demonstrate rapid, transient effects on the modulation state (defined as the fraction of the enzyme present in the catalytically active form) of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase, E.C. 1.1.1.34). Insulin elevated, while glucagon, cAMP or cGMP lowered HMG-CoA reductase modulation state within 10 to 15 min. These changes were accompanied by a parallel change in sterol synthesis. Total HMG-CoA reductase activity was not altered. Rapid modulation of HMG-CoA reductase activity therefore constitutes a viable in vivo control mechanism. By contrast to the hormones and second messengers, mevalonolactone lowered both HMG-CoA reductase modulation state and total reductase quantity.     

Altered activation state of hydroxymethylglutaryl-coenzyme A reductase in liver tumors

Extensive studies have demonstrated that the normal inhibition of cholesterol synthesis by cholesterol feeding is decreased in all hepatomas studied in vivo. This loss of the normal feedback regulation of cholesterol synthesis has been shown to be due to the failure of cholesterol ingestion to inhibit the activity of hydroxymethylglutaryl (HMG)-CoA reductase. The basis for this absence of feedback control of cholesterogenesis is unknown. Studies to date have not demonstrated structural or kinetic differences between the HMG-CoA reductase of normal liver and hepatoma. The present study, however, demonstrates significant differences in the activation state of HMG-CoA reductase from normal liver and hepatoma. In normal liver only approximately 10-20% of the microsomal HMG-CoA reductase is in the dephosphorylated, active form while 80-90% is in the phosphorylated, inactive state. In contrast, in three different Morris hepatomas in vivo, from 53 to 73% of the HMG-CoA reductase is in the active state. That the increased activation state in hepatomas is a property of tumor tissue and is not solely due to rapid growth is demonstrated by the fact that in both fetal and regenerating liver an enhanced activation state of HMG-CoA reductase is not observed. Additionally, preincubation with magnesium and ATP results in the inhibition of HMG-CoA reductase both in tumor and in liver. Presumably, this decrease in HMG-CoA reductase activity is due to the phosphorylation of the enzyme. Similarly, the preincubation of tumor and liver microsomes with phosphatase results in an increase in HMG-CoA reductase activity presumably by the dephosphorylation of the enzyme to its active form. The relationship between the altered activation state of HMG-CoA reductase in hepatomas and the reduction in the feedback regulation of this enzyme in liver tumors remains to be explored.     

Altered kinetic properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase following dietary manipulations

The microsomal enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the rate-limiting step in the cholesterogenic pathway and was proposed to be composed in situ of 2 noncovalently linked subunits (Edwards, P.A., Kempner, E.S., Lan, S.-F., and Erickson, S.K. (1985) J. Biol. Chem. 260, 10278-10282). In the present report, the activities and kinetic properties of HMG-CoA reductase in microsomes isolated from livers of rats fed on diets supplemented with either ground Amberlite XAD-2 ("X"), cholestyramine/mevinolin ("CM"), or unsupplemented, normal rat chow ("N"), were compared. The specific activities of HMG-CoA reductase in X and CM microsomes were, respectively, 5- and 83-fold higher than that of N microsomes. In NADPH-dependent kinetics of HMG-CoA reductase activated with 4.5 mM GSH, the concentration of NADPH required for half-maximal velocity (S0.5) was 209 +/- 23, 76 +/- 23, and 40 +/- 4 microM for the N, X, and CM microsomes, respectively. While reductase from X microsomes displays cooperative kinetics toward NADPH (Hill coefficient (nH) = 1.97 +/- 0.07), the enzyme from CM microsomes does not (nH = 1.04 +/- 0.07). Similarly to HMG-CoA reductase from CM microsomes, the freeze-thaw solubilized enzyme ("SOL") displays no cooperativity toward NADPH and its Km for this substrate is 34 microM. At 4.5 mM GSH, HMG-CoA reductase from X, CM, and SOL preparations has a similar Km value for [DL]-HMG-CoA, ranging between 13-16 microM, while reductase from N microsomes had a higher Km value (42 microM) for this substrate. No cooperativity towards HMG-CoA was observed in any of the tested enzyme preparations. Immunoblotting analyses of the different preparations demonstrated that the observed altered kinetics of HMG-CoA reductase in the microsomes is not due to preferential proteolytic cleavage of the native 97-100 kDa subunit of the enzyme to the noncooperative 50-55 kDa species. Moreover, it was found that the ratio enzymatic activity/immunoreactivity of the reductase increased in the order N less than X less than CM approximately equal to SOL, indicating that the activity per reductase molecule increases with the induction of the enzyme. These results are compatible with a model suggesting that dietary induction of hepatic HMG-CoA reductase may change the state of functional aggregation of its subunits.     

Inhibition of human leukocyte 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by ascorbic acid. An effect mediated by the free radical monodehydroascorbate

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in microsomes isolated from cultured lymphoid (IM-9) cells or freshly isolated human leukocytes was markedly decreased by either ascorbic acid or its oxidized derivative, dehydroascorbate. Inhibition of IM-9 leukocyte HMG-CoA reductase activity was log linear between 0.01 and 10 mM ascorbic acid (25 and 81% inhibition, respectively) and 0.1 and 10 mM dehydroascorbate (5 and 75% inhibition, respectively). Inhibition was noncompetitive with respect to HMG-CoA (Km = 10.2 microM (RS); ascorbic acid, Ki = 6.4 mM; dehydroascorbate, Ki = 15 mM) and competitive with respect to NADPH (Km = 16.3 microM; acetic acid, Ki = 6.3 mM; dehydroascorbate, Ki = 3.1 mM). Ascorbic acid and dehydroascorbate are interconverted through the free radical intermediate monodehydroascorbate. Reducing agents are required to convert dehydroascorbate to monodehydroascorbate, but prevent formation of the free radical from ascorbate. In microsomes from IM-9 cells, the reducing agent, dithiothreitol, abolished HMG-CoA reductase inhibition by ascorbate but enhanced inhibition by dehydroascorbate. In addition, the concentration of monodehydroascorbate present in ascorbate solutions was directly proportional to the degree of HMG-CoA reductase inhibition by 1.0 mM ascorbate. Fifty per cent inhibition of enzyme activity occurred at a monodehydroascorbate concentration of 14 microM. These data indicate that monodehydroascorbate mediates inhibition of HMG-CoA reductase by both ascorbate and dehydroascorbate. This effect does not appear to be due to free radical-induced membrane lipid modification, however, since both ascorbate and dehydroascorbate inhibited the protease-solubilized, partially purified human liver enzyme. Since inhibition of HMG-CoA reductase occurs at physiological concentrations of ascorbic acid in the human leukocyte (0.2-1.72 mM), this vitamin may be important in the regulation of endogenous cholesterol synthesis in man.     

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.     

Acute effects of starvation and treatment of rats with anti-insulin serum, glucagon and catecholamines on the state of phosphorylation of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase in vivo.

The fraction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in the dephosphorylated (active) form in rat liver in vivo was measured after various experimental treatments of animals. Intraperitoneal injection of glucose (to raise serum insulin concentrations) into rats 4 h into the light phase (L-4) resulted in a transient (30 min) increase in the expressed (E)/total (T) activity ratio of HMG-CoA reductase without any change in total activity (obtained after complete dephosphorylation of the enzyme). Conversely, intravenous injection of guinea-pig anti-insulin serum into rats 4 h into the dark phase (D-4) significantly depressed the E/T ratio within 20 min. Intravenous injection of glucagon into normal rats at this time point did not affect the degree of phosphorylation of the enzyme, in spite of a 10-fold increase in hepatic cyclic AMP concentration induced by the hormone treatment. A 3-fold increase in the concentration of the cyclic nucleotide induced by adrenaline infusion was similarly ineffective in inducing any change in expressed or total activities of hepatic HMG-CoA reductase. However, when insulin secretion was inhibited, either by the induction of streptozotocin-diabetes or by simultaneous infusion of somatostatin, glucagon treatment was able to depress the expressed activity of HMG-CoA reductase (i.e. it increased the phosphorylation of the enzyme). Therefore insulin appears to have a dominant role in the regulation of the phosphorylation state of hepatic HMG-CoA reductase. In apparent corroboration of this suggestion, short-term 4 h food deprivation of animals before D-4 resulted in a marked decrease in the E/T activity ratio of reductase, which was not affected further by an additional 8 h starvation. By contrast, the total activity of the enzyme was not significantly affected by 4 h starvation, but was markedly diminished after 12 or 24 h starvation. Longer-term starvation also produced a chronic increase in the degree of phosphorylation of the enzyme. These results are discussed in relation to the role of reversible phosphorylation in the control of hepatic HMG-CoA reductase activity in vivo.     

Solubilization and partial purification of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase

This paper describes an effective method for the solubilization of microsomal HMG-CoA reductase from rat liver. Exposing the microsomes to a freeze-thaw treatment solubilized 80% of the microsomal reductase activity. Subsequently, a 25-fold purification has led to an enzyme preparation with a specific activity of 10–14 nmoles MVA per min per mg of protein and an increased stability.     

Cholesterol-mediated regulation of HMG-CoA reductase in microsomes from human skin fibroblasts and rat liver

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was determined in microsomes from human skin fibroblasts and rat liver that had been variously manipulated in vivo or in tissue culture to up- and down-regulate the enzyme. The cholesterol content of these microsomal preparations was then altered by depletion to or enrichment from either cholesterol-free or cholesterol-rich lipid vesicles. Microsomes from human skin fibroblasts responded to cholesterol depletion by increasing HMG-CoA reductase activity and by decreasing it in response to cholesterol enrichment. This was independent of the initial enzyme activity or the tissue culture conditions. Alterations in cholesterol content of rat liver microsomes in vitro failed to demonstrate any significant changes in HMG-CoA reductase activity whether the microsomes started with low enzyme activity (cholesterol-fed rats) or with high enzyme activity (cholestyramine-treated rats). The results are discussed in relation to previously published data and in respect to differences in the control of the human skin fibroblast and rat liver enzymes.     

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.