Cholesterol Biosynthesis In Liver Research Articles

Investigations on the mechanism of the hypocholesterolemic action of 1-ethoxysilatrane

Intraperitoneal administration of the nontoxic silicon compound, 1-ethoxysilatrane, to the rat did not cause proliferation of hepatic mitochondria or of endoplasmic reticulum, nor did it affect mitochondrial oxidative phosphorylation. The activities of cholesterol 7 alpha-hydroxylase in hepatic microsomes and of cholesterol oxidase in mitochondria respectively were unaffected by silatrane treatment. The rate of release of bile, whose composition remained unchanged, also was not increased in silatrane-treated animals. The results indicated that the compound did not affect the pathway of cholesterol degradation. A progressive decrease in the activity of hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase was observed on administration of the compound over a period of three weeks. Consistent with this, cholesterol biosynthesis in liver as measured by incorporation of radioactive precursors, acetate and water but not mevalonate, was significantly decreased in silatrane-treated animals. However, enzyme-linked immunosorbent assay revealed that the concentration of HMGCoA reductase protein was not decreased by the treatment indicating that inactivated enzyme was also present in such microsomes. Addition of silatrane to microsomes in the assay system did not cause inhibition indicating that the inactivation is by an indirect mechanism. It is concluded that the hypocholesterolemic action of the compound rested entirely on the inhibition of cholesterol biosynthesis in vivo by inactivation of the rate-limiting enzyme HMGCoA reductase.

Effect of Shosaikoto, Daisaikoto and Sannoshashinto (traditional Japanese and Chinese medicines) on experimental hyperlipidemia in rats

Effect of Sannoshashinto, Shosaikoto and Diasaikoto, Japanese and Chinese traditional medicinal mixtures (kampohozai), on cholesterol-induced hypercholesterolemia, aging-induced hyperlipidemia and cholesterol turnover were studied in rats. Sannoshashinto, Shosaikoto and Daisaikoto reduced the hypercholesterolemia induced by a high cholesterol diet and Sannoshashinto and Daisaikoto improved the atherogenetic index. Liver total cholesterol as increased by a high cholesterol diet was reduced by all three kampohozai. Furthermore, the increases of serum and liver triglyceride were also inhibited. In an experiment using untreated aging rats, both serum total cholesterol and serum triglyceride levels were increased. The aging-induced increases of serum total cholesterol were inhibited by Sannoshashinto and Daisaikoto and the increases of serum triglyceride were reduced by all three kampohozai. These drugs showed no effect on cholesterol biosynthesis in liver. Sannoshashinto, however, appeared to accelerate the disappearance of cholesterol from blood, while Daisaikoto inhibited the cholesterol absorption from the intestine.

Effects of phenotype, sex, and diet on plasma lipids in LA/N-cp rats.

The LA/N-corpulent (cp) rat is a recently developed congenic strain which exhibits obesity. The effects of phenotype and sex on serum and lipoprotein lipid content were examined in LA/N-cp rats fed either a control or an atherogenic diet high in saturated fat and protein. Obese rats were pair-fed to equivalent lean animals. Results from this study indicate that sex, phenotype, and diet exert significant effects on plasma and lipoprotein cholesterol content. Plasma cholesterol levels were higher in obese compared with lean rats, females than in males, and rats consuming the atherogenic diet compared with the control diet. Plasma and lipoprotein triglyceride levels were significantly increased only in obese compared with lean animals. The increased plasma cholesterol and triglyceride was observed primarily in the chylomicron and very low density lipoprotein fractions. Increased levels of plasma cholesterol were not a result of increased dietary cholesterol absorption or increased liver cholesterol biosynthesis. These data suggest that LA/N-cp rats can serve as a unique rodent model for the study of the interrelationships between hyperlipidemia, obesity, and coronary heart disease.

Inhibitory effect of a new mycotic agent, piritetrate on ergosterol biosynthesis in pathogenic fungi.

A new antimycotic agent piritetrate, a thiocarbamate derivative, was found to interfere with fungal sterol biosynthesis. Sterol biosynthesis was measured by means of incorporation of radioactivity from [14C]acetate into individual sterol fractions and their precursor compounds extracted from cells of Candida albicans, Cryptococcus neoformans, and Trichophyton mentagrophytes. Piritetrate was a much more potent inhibitor of fungal squalene epoxidation than a related drug, naphthiomate. The greater antifungal efficacy of the former was reflected in its greater inhibitory action on sterol biosynthesis than the latter. Such inhibitory effects were also demonstrated in in vitro labelling experiments with [14C]mevalonate on the S10 fraction (10,000 g supernatant of homogenate) of C. albicans cells. At 5 x 10(-7) M concentration, piritetrate gave almost 90% inhibition of ergosterol biosynthesis in the S10 fraction of C. albicans cells. Furthermore, this agent was highly specific for fungal enzymes involved in ergosterol biosynthesis, with no detectable effects on mammalian cholesterol biosynthesis in rat liver at the therapeutic concentration.

Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver.

We have used isopycnic density gradient centrifugation to study the distribution of several rat liver microsomal enzymes of cholesterol synthesis and metabolism. All of the enzymes assayed in the pathway from lanosterol to cholesterol (lanosterol 14-demethylase, steroid 14-reductase, steroid 8-isomerase, cytochrome P-450, and cytochrome b5) are distributed in both smooth (SER) and rough endoplasmic reticulum (RER). The major regulatory enzyme in the pathway, hydroxymethylglutaryl-CoA reductase, also was found in both smooth and rough fractions, but we did not observe any associated with either plasma membrane or golgi. Since cholesterol can only be synthesized in the presence of these requisite enzymes, we conclude that the intracellular site of cholesterol biosynthesis is the endoplasmic reticulum. This is consistent with the long-held hypothesis. When the overall pathway was assayed by the conversion of mevalonic acid to non-saponifiable lipids (including cholesterol), the pattern of distribution obtained in density gradients verified its general endoplasmic reticulum localization. The enzyme acyl-CoA-cholesterol acyltransferase which removes free cholesterol from the membrane by esterification, was found only in the rough fraction of endoplasmic reticulum. In addition, when the RER was degranulated by the addition of EDTA, the activity of acyl-CoA-cholesterol acyltransferase not only shifted to the density of SER but was stimulated approximately 3-fold. The localization of these enzymes coupled with the stimulatory effect of degranulation on acyl-CoA-cholesterol acyltransferase activity has led us to speculate that the accumulation of free cholesterol in the RER membrane might be a driving factor in the conversion of RER to SER.

The regulation of acetoacetyl-CoA synthetase activity by modulators of cholesterol synthesis in vivo and the utilization of acetoacetate for cholesterogenesis.

[3-14C]Acetoacetate, injected subcutaneously into rats, was rapidly incorporated into hepatic cholesterol and fatty acids. Injection of radiolabeled acetoacetate, acetate, or glucose resulted in the preferential incorporation of acetoacetate into cholesterol. The postmitochondrial supernatant of a rat liver homogenate has the capacity to synthesize radiolabeled sterols from [3-14C]acetoacetate, and this capacity surpasses its capacity to utilize [1-14C]acetate. The activity of acetoacetyl-CoA synthetase, a cytoplasmic enzyme that activates acetoacetate, was found to be highly regulated by modulators known to affect the activity of 3-hydroxy-3-methylglutaryl-CoA reductase and/or cholesterol biosynthesis in liver and adrenals of adult rats. Acetoacetyl-CoA synthetase activity was depressed by feeding cholesterol or mevalonate administration and was enhanced by the addition to the diet of mevinolin and/or cholestyramine. The activity of acetoacetyl-CoA synthetase in adrenals was enhanced by treatment of the animals with 4-aminopyrazolopyrimidine. These changes in activity of acetoacetyl-CoA synthetase were in synchrony with changes in the activities of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase. The evidence suggests that the regulation of acetoacetyl-CoA synthetase activity and the utilization of acetoacetate for lipogenesis is closely linked to the regulation of cholesterogenesis.

Cholesterol and bile-acid metabolism in normal and cholesterol-fed rabbits after partial lleal bypass

The effects of partial ileal bypass on plasma lipids, tissue cholesterol biosynthesis, and liver 7α-hydroxylation were studied in rabbits on a normal diet and after cholesterol feeding. The surgical procedure was minimally effective in control animals, whereas it dramatically reduced (even below baseline) plasma cholesterol levels in the diet-treated group. Liver cholesterol biosynthesis from [ 14C]acetate was not detectable in control rabbits. In these, biosynthesis from [ 14C]mevalonate was revealed in the liver and intestine, particularly in the duodenum. Cholesterol feeding totally suppressed the intestinal and liver biosynthetic activity, both from [ 14C]acetate and [ 14C]mevalonate. Partial ileal bypass significantly raised liver cholesterol biosynthesis from [ 14C]mevalonate, particularly in animals on the hyper-cholesterolemic regimen. No cholesterol 7α-hydroxylase activity, as determined by a mass fragmentographic technique, was found in control and cholesterol-fed rabbits; after partial ileal bypass, activities of 167 and 643 pmol·mg protein −1·hr −1, respectively, were found in controls and hypercholesterolemic animals. These findings underline the sensitivity of rabbits both to cholesterol feeding and to partial ileal bypass, a condition possibly related to the very low sterol synthetic activity in these animals. They also suggest that the surgical procedure may alter some regulatory step in tissue cholesterol biosynthesis.

Cholesterol biosynthesis in polychlorinated biphenyl-treated rats

After administration of polychlorinated biphenyls (PCB) at 0.05% (w/w) of the diet to Wistar rats for 30 days, followed by intraperitoneal injection of tritiated water, [ 14C]mevalonate, and [ 14C]acetate, there was a decrease in cholesterol biosynthesis in rat liver. No significant change in cholesterol formation was observed when PCB was administered at 0.01% (w/w) of the diet. In vitro inhibition of cholesterol synthesis by rat liver microsomes was observed with PCB. Squalene 2,3-oxidocyclase activity of rat liver microsomes was not significantly altered. Desmosterol δ 24 reductase activity was inhibited only at relatively high concentrations of PCB. There was increased incorporation of radioactivity into squalene and lanosterol, in vitro, in the presence of PCB. The primary inhibition of cholesterol biosynthesis appears to be at the demethylation and rearrangement reactions between lanosterol and cholesterol in the biosynthetic pathway.

Biochemical controls of liver cholesterol biosynthesis

Major endogenous regulatory controls of cholesterol synthesis including dietary-feedback inhibition, circadian rhythms, feeding/fasting fluctuations, and enterohepatic bile acid circulation are discussed in terms of biochemical control mechanisms of liver cholesterol biosynthesis. Other likely control mechanisms such as cofactors, enzyme levels, and enzyme activities are noted. The current state of the biochemical knowledge is very dependent upon integrated data from one experimental animal - the rat. Inferences and implications in the human are thus limited.

Effects of portacaval shunt and transposition on fatty acid and cholesterol biosynthesis in rat liver.

Male rats underwent either portacaval shunt or portacaval transposition; in both cases, sham-operated pair-fed rats served as controls. Three weeks after a portacaval shunt, fasting serum values of glucose (-35%) and cholesterol (-24%) were lower, and fasting plasma glucagon was higher (+65%). The wet weight of the liver and its total content in DNA, RNA, and protein decreased by 43, 40, 43, and 48%, respectively. The supernatant of liver obtained after centrifugation at 700 g incorporated less [1-14C]acetate (-56%) into fatty acids and less [1-14C]acetate (-94%) and [2-14C]mevalonate (-37%) into cholesterol. The activity of acetyl CoA carboxylase was reduced by 56%. The in vivo incorporation of [3H]H2O into liver fatty acids was 83% lower and that into liver cholesterol was 39% lower than in pair-fed controls. Several of the preceding parameters, including in vitro and in vivo labeling of hepatic fatty acids and cholesterol, were found to be mostly normal in rats with portacaval transposition. These data suggest that the reduction of fatty acid and cholesterol biosynthesis in the liver of rats with portacaval shunt was due to the reduction of total hepatic blood flow rather than to the diversion of portal blood constituents.

Pig liver phosphomevalone kinase. 1. Purification and properties.

Pig liver phosphomevalonate kinase (EC 2.7.4.2) has been purified to homogeneity as shown by polyacrylamide gel electrophoresis. The molecular weight estimates range from 21,000 to 22,500. Each molecule is composed of one polypeptide chain. The presence of SH-containing reagents is essential for the preservation of enzymes activity at all steps in the purification. The enzyme shows absolute specificity for ATP and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 7.5 to over 9.5. Kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.075 mM and 0.46 mM have been obtained for phosphomevalonate and ATP, respectively. Amino acid composition shows a high content of acid amino acids, one cysteine residue per molecule of enzyme, and the absence of methionine. The results obtained suggest that the enzyme plays no regulatory function in cholesterol biosynthesis in pig liver, although a variable enzyme content was detected in different livers.

Mode of action of the lipid-lowering agents, clofibrate and BM 15075, on cholesterol biosynthesis in rat liver

When rats were fed a diet containing 0.3% clofibrate or a derivative of this drug, BM 15075, serum cholesterol was lowered within 3–7 days by 26–38%. Both drugs diminished the activity of hydroxymethylglutaryl CoA reductase, the regulatory enzyme of hepatic cholesterol biosynthesis, in rat liver microsomes by about 60% under the same conditions. The decrease in the activity of the enzyme obviously is due to changes in the amount of enzyme protein. Under in vitro conditions, microsomal hydroxymethylglutaryl CoA reductase was inhibited competitively by (1.35 mM) clofibric acid (sodium salt) and by BM 15075 (1 mM) with respect to its substrate. These results give evidence that these drugs can affect both, the rate of synthesis and the substrate affinity of hydroxymethylglutaryl CoA reductase.

L-histidine-induced suppression of lipogenic enzymes

Diets supplemented 5% with L-histidine produce hypercholesterolemia and increase cholesterol biosynthesis in rat liver. We now report an inhibitory effect of L-histidine on lipogenic enzymes in the liver. In this study, L-histidine was added to chow and fat-free diets and fed to rats for 18 days. After two days of fasting, the rats were refed the same diet for three days prior to sacrifice. L-histidine decreased fatty acid synthetase activity by 51% when it was added to the chow diet and by 26% when it was added to the fat-free diet. Isocitrate dehydrogenase activity was not altered significantly in rats fed diets supplemented with L-histidine.

Hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity in hamsters on a lithogenic diet.

The activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme for liver cholesterol biosynthesis, has been determined in young hamsters given a diet known to produce cholesterol gallstones in this species and compared to the activity found in chow-fed hamsters. None of the hamsters fed the lithogenic diet for 15 days or less developed gallstones but 74 percent of those on the diet for 26 to 49 days had cholesterol gallstones. None of the chow-fed animals developed gallstones. The mean HMG CoA reductase activity of hamsters on the lithogenic diet at 4 to 6 hours after the onset of the dark period was 20 times greater than in hamsters in the fasting state and 12 times that of fed hamsters on the chow diet. These greatly elevated enzyme activities were found as early as 2 to 15 days on the lighogenic diet and thus long before the gallstones appeared. The percentage of the bile acid pool represented by chenodeoxycholic acid was significantly higher, and that cholic acid correspondingly lower on the gallstone-inducing diet. This shift in bile acid composition may have contributed to gallstone formation. No significant accumulation of cholesterol was noted in liver or carcass. Increased synthesis of cholesterol induced by the greatly stimulated enzyme activity in hamsters on the lithogenic diet could account for the increased secretion of cholesterol in bile noted by others with subsequent supersaturation of gallbladder bile with respect to cholesterol, and then gallstone formation.

Sterol carrier protein hypothesis: Requirement for three substrate-specific soluble proteins in liver cholesterol biosynthesis

The 105,000 x g supernatant (S 105) of liver is required for the conversion of squalene to cholesterol by microsomal membranes. Substantial controversy has existed concerning the properties of what was originally considered to be a single sterol carrier protein present in S 105 and required for this conversion. We have now resolved this controversy by the discovery that S 105 contains several sterol carrier proteins. Based upon experiments with three substrates, three substrate-specific soluble proteins (with different properties) have been identified which operate at distinct points in microsomal cholesterol synthesis. These proteins are provisionally designated sterol carrier protein 1 (SCP 1), sterol carrier protein 2 (SCP 2), and sterol carrier protein 3 (SCP 3). SCP 1 is required for the microsomal conversion of squalene to lanosterol, SCP 2 for the microsomal conversion of 4,4-dimethyl-Δ 8-cholesterol to C 27-sterols, and SCP 3 for the microsomal conversion of 7-dehydrocholesterol to cholesterol. Available evidence is consistent with the proposal that a given sterol carrier protein is a soluble constituent of a single microsomal enzyme or enzyme complex, and that it participates both as a carrier for the water-insoluble substrate and as an essential enzyme constituent facilitating catalysis. It may well be that enzymatic transformations of water-insoluble substrates require both microsomal membranes and substrate-specific soluble proteins. This requirement could be a common biological mechanism for water-insoluble substrates.

Effect of cholesterol feeding on cholesterol biosynthesis in maternal and foetal rat liver.

Hepatic cholesterol biosynthesis has been studied in rat foetuses whose mothers had been fed on a cholesterol rich diet during the last week of gestation. Foetal liver was found to be capable of synthesizing cholesterol from acetate in vitro. The rate of incorporation of labelled acetate into digitonin precipitable sterols, fatty acids and CO(2) in foetal liver was much higher than that found in maternal liver. Cholesterol feeding reduced the rate of sterol synthesis in maternal liver but it did not have any appreciable effect on foetal liver. In order to investigate whether this lack of feed-back control in foetal liver could be attributable to an obstacle to the placental transfer of dietary cholesterol. 14-C-cholesterol was administered to the pregnant rats and its distribution in maternal and foetal liver and plasma was studied. Our results indicate that placental transfer of cholesterol from mother to foetus occurs very slowly so that only a small proportion of labelled cholesterol is found in foetal plasma over a 48 hour period following the administration of radioactive cholesterol. Cholesterol transferred from the mother into the foetal plasma is efficiently taken up by the foetal liver. These findings would suggest that the low amount of dietary cholesterol transferred from the mother into the foetal plasma is not sufficient to activate the control mechanism of the cholesterol biosynthetic pathway in the foetal liver.

Compartmentation of the early steps of cholesterol biosynthesis in mammalian liver.

Cholesterol synthesis was studied in the isolated perfused rat liver and with cell-free preparations by incorporation measurements of3H from3HOH and of carbon label from [1-14C]-acetate. Using specific inhibitors such as (-)-hydroxycitrate, kynurenate, and avidin the following conclusions were reached:

Effect of prostaglandin E1 on cholesterol biosynthesis in rat liver.

La prostaglandina E1 (PGE1) ha un duplice effetto sulla sintesi epatica del colesterolo: a basse concentrazioni determina un incremento della incorporazione di acetato in colesterolo, mentre a concentrazini superiori a 50 nmoli essa determina una riduzione di questo paramentro.

Mechanism of squalene cyclization: The chiral origin of the C-7 and C-15 hydrogen atoms of fusidic acid

The C-2 protons of mevalonic acid are incorporated into C-7 and C-15 of fusidic acid with retention of their stereochemical integrity; this finding is in agreement with the accepted mode of squalene-oxide cyclization to the C-20 protosterol cation, and supports the proposed mechanisms for the enzymatic events at C-7 and C-15 in the biosynthesis of cholesterol in rat liver.

3-Hydroxy-3-methylglutaryl Coenzyme A Reductase: SOLUBILIZATION AND PURIFICATION OF A COLD-SENSITIVE MICROSOMAL ENZYME

Abstract 3-Hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme of cholesterol biosynthesis in rat liver, has been solubilized and purified 130-fold from rat liver microsomes. Evidence is presented to suggest that the enzyme is localized to the surface of the microsomal membrane. 3-Hydroxy-3-methylglutaryl-CoA reductase can be removed from microsomes by mild treatments such as exposure to high salt or glycerol concentrations which leave the bi-laminar structure of the microsomal membrane intact. The solubilized enzyme is rapidly inactivated by chilling to 4° and is protected from this cold-induced inactivation by salt only at concentrations in the range of 4 m. 3-Hydroxy-3-methylglutaryl-CoA reductase is the first solubilized microsomal enzyme reported to be cold-sensitive.