Evolution of 3-hydroxy-3-methylglutaryl-CoA reductase and microsomal membrane fluidity throughout chick embryo development

The pattern of chick liver and brain 3-hydroxy-3-methylglutaryl-CoA reductase and its relationship with changes in microsomal membrane fluidity was studied during embryonic and postnatal development. A peak of brain activity was found at 19 days of embryonic development, while liver activity only increased after hatching. A significant increase in cholesterol content of brain microsomes occurred at about 14 days of incubation, decreasing afterwards. No significant variations were observed in liver microsomes during the same period. A similar profile was found in the phospholipid content of both brain and liver microsomes. The cholesterol/lipidic phosphorus molar ratio of brain and liver microsomes did not exhibit significant changes throughout embryonic and postnatal development. These results demonstrate that membrane-mediated control does not regulate the evolution of reductase activity during this developmental period.     

Early effects of dietary orotic acid upon liver lipid synthesis and bile cholesterol secretion in rats

Dietary orotic acid is known to cause impaired fatty acid synthesis and increased cholesterol synthesis in rats. We found that the impaired fatty acid synthesis occurs during the first day of orotic acid feeding and, in studies with albumin-bound [1-14C]palmitic acid, an associated decrease in the rate of esterification of this fatty acid into triacylglycerol, phospholipid, and cholesteryl ester was observed. These changes may result from the known decreases in liver levels of adenine nucleotides or, as reported here, from decreased liver CoASH levels in orotic acid-fed rats. The increase in hepatic cholesterol synthesis occurred during the second day of orotic acid feeding. It was detected by increased incorporation of [1,2-14C]acetate into cholesterol by liver slices and by a 7-fold increase in HMG-CoA reductase activity. At the same time the biliary output of cholesterol was increased 2-fold and studies using 3H2O revealed that the output of newly synthesized cholesterol in bile was increased 5-fold. The content of cholesteryl ester in hepatic microsomes decreased during orotic acid feeding but free cholesterol was unchanged. The findings are interpreted to suggest that the increased bile cholesterol secretion caused by orotic acid is a result of impaired hepatic cholesterol esterification and that the increase in HMG-CoA reductase activity is a result of diminished negative feedback due to the depleted content of cholesteryl ester in the hepatic microsomes.     

Hypolipidemic activity of dipyridamole: effects on the main regulatory enzyme of cholesterogenesis.

The in vivo dipyridamole treatment for 16 days produced a significant decrease in chick plasma cholesterol, mainly due to the esterified form. This effect was especially patent in the VLDL + LDL fraction. Similar results were observed in triglyceride content. To our knowledge, this is the first report on this hypolipidemic effects of dipyridamole. Total and esterified cholesterol increased after the same treatment in chick liver, while brain cholesterol content was not affected. Hepatic 3-hydroxy-3- methylglutaryl-CoA reductase activity was drastically reduced, while other secondary regulatory enzymes such as mevalonate kinase, mevalonate 5-phosphate kinase and mevalonate 5-pyrophosphate decarboxylase did not change significantly. No significant differences were found in cholesterol and lipidic phosphorus from liver microsomes, so that the effect of dipyridamole on reductase activity cannot be due to modifications in cholesterol/lipidic phosphorus molar ratio. Neither of these enzyme activities was affected in vitro by dipyridamole.     

In vitro modification of cholesterol content of rat liver microsomes. Effects upon membrane 'fluidity' and activities of glucose-6-phosphatase and fatty acid desaturation systems

The cholesterol content of rat liver microsomal membranes was modified in vitro by incubating microsomes and cytosol with liposomes prepared by sonication of microsomal lipids and cholesterol. In this way, the cholesterol to phospholipid molar ratio was increased from 0.11-0.13 in untreated microsomes to a maximal of 0.8 in treated ones. Cholesterol incorporation in microsomes produced an increase in the diphenyl-hexatriene steady-state fluorescence anisotropy and a decrease in the efficiency of pyrene-excimer formation which indicated a decrease in the rotational and translational mobility, respectively, of these probes in the membranes lipid phase. Cholesterol incorporation in microsomes did not affect significantly the glucose-6-phosphatase activity in 0.1% Triton X-100 totally disrupted microsomes, but diminished the glucose-6-phosphatase activity of 'intact' microsomes. This indicates that possibly the glucose 6-phosphate translocation across the microsomal membrane is impeded by an increase in the membrane apparent 'microviscosity'. Cholesterol incorporation in microsomes decreased NADH-cytochrome c reductase without affecting NADH-ferricyanide reductase activity. The delta 9 desaturation reaction rate was enhanced by cholesterol incorporation at low but not at high palmitic acid substrate concentration. delta 5 and delta 6 desaturase reaction-rates were increased both at low and high fatty acid substrate concentrations. These results suggest that a mechanism involving fatty acid desaturase enzymes, might exist to self-regulate the microsomal membrane lipid phase 'fluidity' in the rat liver.     

Independent regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and chylomicron remnant receptor activities in rat liver.

Treatment of rats with pharmacological doses of oestrogen resulted in a 3-fold decrease in the activity of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) and a 4-fold increase in saturable binding of 125I-labelled chylomicron remnants to liver membranes in vitro. Intragastric administration of mevalonolactone to rats did not affect the capacity of the liver membranes to bind to labelled chylomicron remnants even though there was a substantial decrease in the activity of HMG-CoA reductase. Similar results were obtained after cholesterol feeding. Simultaneous treatment of rats with cholestyramine and compactin increased hepatic HMG-CoA reductase activity 6-fold. However, liver membranes derived from these animals showed no change in their capacity to bind to labelled chylomicron remnants in vitro. Administration of mevalonolactone to the cholestyramine/compactin-treated animals also failed to produce a change in remnant-binding capacity. Although administration of mevalonolactone alone produced a significant 3-fold decrease in the activity of hepatic HMG-CoA reductase it was unable to suppress significantly the increase in enzyme activity caused by treatment with cholestyramine and compactin.     

Alterations of rat hepatic cholesterogenesis by heterologous lipoproteins.

Heterologous human lipoproteins were infused into rats in order to change acutely the lipoprotein pattern to a predominant kind and the effect on hepatic cholesterogenesis was subsequently observed. A 4-h intravenous infusion of human low density and very low density lipoproteins into rats produced a significant decrease in the incorporation of acetate into cholesterol in both liver slices and homogenates. An infusion of similar concentrations of human high density lipoprotein produced a significant increase in hepatic cholesterol synthesis. These infusions did not change mevalonate conversion to cholesterol in either the homogenates or slices. Concomitant with the changes in hepatic cholesterol synthesis were changes of similar magnitudes in the activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase. These alterations in hepatic cholesterol synthesis were associated with significant changes in microsomal cholesterol content. There was a significant increase in hepatic cholesterol synthesis with the infusion of apoproteins of high density lipoprotein. The apoproteins of very low density lipoprotein had no effect on hepatic cholesterogenesis. These studies indicate that circulating lipoproteins modify hepatic cholesterol synthesis and that the apoproteins of these lipoproteins may themselves be important for this action.     

Differential response of chick liver and brain membranes to short ethanol treatment

The effect of 60 hr ethanol ingestion on lipid composition of liver and brain membranes from 2-day-old chicks was investigated. Analysis of hepatic membrane cholesterol shows that ethanol induced a slight increase in microsomes exclusively due to free cholesterol while mitochondria was not affected. In brain, both fractions showed a clear increase in their cholesterol content, while a high decrease was observed in myelin. Free cholesterol was also the main responsible for the changes found in brain. The ethanol-treated animals showed an alteration in their phospholipid composition exclusively in brain microsomes and myelin. Despite all these changes, the values of cholesterol/phospholipid molar ratio in both liver and brain membranes remained unaltered after short ethanol treatment. Our results indicate that neonatal chick brain membranes appears to be especially sensitive to the presence of ethanol.     

Correlation between changes in membrane lipid composition induced by dietary lipid and membrane-bound enzyme activity in chick liver

The activity of acyl-CoA: cholesterol acyltransferase in the liver-microsomal fraction was considerably reduced in chicks fed on diet containing unsaturated fat, whereas the activity of HMG-CoA reductase and NADPH cytochrome c reductase was not affected. The fatty acid composition of the microsomes was modified appreciably by this dietary condition and there was no change in the phospholipid or cholesterol levels. The addition of cholesterol to the fat supplemented diet resulted in a considerable increase in the microsomal cholesterol content. A decrease in HMG-CoA reductase and an increase ACAT activity was observed compared with the corresponding values from both the groups fed on a standard diet and a fat supplemented diet with no cholesterol. These results suggest that acyl-CoA: cholesterol acyltransferase is modulated by alteration in the fatty acid composition of the microsomal membrane, while the cholesterol content of the microsomes shows a close relationship with the HMG-CoA reductase activity.     

Alteration of biliary ursodeoxycholic acid in guinea pig during early stages of cholestyramine feeding

The effects of cholestyramine feeding on biliary ursodeoxycholic acid, fecal excretion of bile acids and neutral sterols on cholesterol 7α-hydroxylase and hepatic HMG-CoA reductase were examined in the guinea pig. In the bile there was a 57% decrease in the concentration of ursodeoxycholic acid while an increase was observed in the concentration of chenodeoxycholic acid. Cholestyramine feeding for ten days resulted in a decrease in plasma cholesterol levels and an increase in both hepatic HMG-CoA reductase and cholesterol 7α-hydroxylase activities. The fecal excretion of both bile acids and neutral sterols was significantly increased.     

In vitro regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A: cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine

The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A:cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA:cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and 'reactivation' of enzyme activity. Cytosol caused a 78% increase in acyl-CoA:cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl2 and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A:cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA:cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500 X g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl2, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA:cholesterol acyltransferase activity is observed.     

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

Summary 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 -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 immunosorbant 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.     

Differential effects of dietary fat on chick plasma and liver composition and HMG-CoA reductase activity

The comparative effects of diet supplementation with 10% saturated fat rich in 12:0 and 14:0 fatty acids (coconut oil), without and with 1% added cholesterol, and with 10% unsaturated fat rich in n-3 polyunsaturated fatty acids (menhaden oil) on cholesterol metabolism in neonatal chicks were examined to clarify the different mechanisms of their hyper- and hypolipidemic action. Supplementation of coconut oil produced a significant hypercholesterolemia after 7 days of treatment, with a similar increase in the amount of both free and esterified cholesterol. Supplementation of coconut oil plus cholesterol produced a higher increase of plasma cholesterol levels (approximately two to three times higher than those found with standard diet). However, supplementation of menhaden oil induced a significant decrease in total cholesterol after only 2 weeks of treatment. Levels of plasma triglycerides did not change by coconut oil addition to the diet, but a significant increase was observed after coconut oil plus cholesterol feeding. Menhaden oil produced a transient decrease in plasma triglycerides. Hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity did not change with coconut oil treatment. However, both coconut oil plus cholesterol and menhaden oil supplemented diets drastically decreased reductase activity after 1 week of dietary manipulation. These results show that different nutrients with the same inhibitory effect on reductase activity produced opposite effects on plasma cholesterol content, suggesting the existence of important differences in the regulatory mechanisms implied in cholesterol biosynthesis and its accumulation in plasma.     

Homeostatic restoration of microsomal lipids and enzyme changes in HMG-CoA reductase and Acyl-CoA : cholesterol acyltransferase in chick liver

We have studied the correlation between changes in the lipid composition in chick liver microsomes and the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acyl-CoA : cholesterol acyltransferase (ACAT) by in vivo and in vitro experiments with 21-day-old chicks. A 5% cholesterol diet for 3 hr produced an increase in the microsomal and plasmatic cholesterol content, a decrease in HMG-CoA reductase activity and a concomitant increase in ACAT activity. The effect produced by the short-term treatment virtually disappeared 27 hr after ending the cholesterol diet. In vitro experiments were carried out by using vesicles constituted by phosphatidycholine/cholesterol and phosphatidylcholine.     

Fatty acid composition and properties of the liver microsomal membrane of rats fed diets enriched with cholesterol.

Male rats were fed diets containing olive (OO) or evening primrose (EPO) oil (10% w/w), with or without added cholesterol (1% w/w). After 6-week feeding, the lipid and fatty acid compositions, fluidity, and fatty acid desaturating and cholesterol biosynthesis/esterification related enzymes of liver microsomes were determined. Both the OO and EPO diets, without added cholesterol, increased the contents of oleic and arachidonic acids, respectively, of rat liver microsomes. The results were consistent with the increases in delta 9 and delta 6 desaturation of n-6 essential fatty acids and the lower microviscosity in the EPO group. Dietary cholesterol led to an increase in the cholesterol content of liver microsomes as well as that of phosphatidylcholine (PC). The cholesterol/phospholipid and PC/PE (phosphatidylethanolamine) ratios were also elevated. Fatty acid composition changes were expressed as the accumulation of monounsaturated fatty acids, with accompanying milder depletion of saturated fatty acids in rat liver microsomes. In addition, the arachidonic acid content was lowered, with a concomitant increase in linoleic acid, which led to a significant decrease in the 20:4/18:2 ratio in comparison to in animals fed the cholesterol-free diets. Cholesterol feeding also increased delta 9 desaturase activity as well as membrane microviscosity, whereas it decreased delta 6 and delta 5 desaturase activities. There was a very strong correlation between fluidity and the unsaturation index reduction in the membrane. Furthermore, the activity of hydroxymethylglutaryl-CoA reductase increased and the activity of acyl-CoA:cholesterol acyltransferase decreased in liver microsomes from both cholesterol-fed groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of copper deficiency on rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity

The effect of copper deficiency on hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key enzyme regulating cholesterol biosynthesis, was investigated in the rat. Male weanling rats were fed semipurified diets containing adequate, marginal, or deficient levels of copper for 6 weeks. Two separate studies were conducted; in the first study, animals were fasted 12 hours prior to analysis and in the second study, animals were fed diets ad libitum. Plasma lipid levels, hepatic cholesterol concentrations, and 3-hydroxy-3-methylglutaryl coenzyme A reductase specific activity, total and active, were determined. Consistent with previous findings, plasma total cholesterol and triglyceride levels were significantly elevated in copper-deficient rats. Copper deficiency resulted in a significant decrease in hepatic total cholesterol levels. Total and active levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase in fed animals were elevated twofold with copper deficiency, with the active form of the enzyme constituting approximately 30% of total activity. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in copper-deficient fasted rats was twofold higher than for the fasted adequate animal; however, fasting did result in a 10-fold reduction in hepatic reductase specific activity. These data support the hypothesis that copper deficiency results in a hypercholesterolemic state in the rat associated with increased hepatic cholesterol synthesis.     

The in vivo regulation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase. Phosphorylation of the enzyme as an early regulatory response following cholesterol feeding

Although substantial evidence supports the conclusion that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the major regulatory enzyme in cholesterol biosynthesis, the molecular events involved in the in vivo regulation of this enzyme have remained obscure. In order to study this problem, rats received a single meal consisting of either rat chow or rat chow containing 2% cholesterol. The rats were killed 60 or 120 min after the beginning of feeding, and liver microsomes were prepared by ultracentrifugation. Two phases of inhibition of microsomal HMG-CoA reductase were observed. The first phase of inhibition, observed 60 min after the beginning of cholesterol feeding, was completely reversed by preincubation of the microsomes with purified phosphoprotein phosphatase. The second phase of inhibition, observed 120 min after the beginning of cholesterol feeding, was not reversed by phosphoprotein phosphatase. These results are consistent with the conclusion that phosphorylation of HMG-CoA reductase is the first step in a series of in vivo regulatory events which produce inactivation and ultimately degradation of the enzyme.     

In vitro regulation of 3-hydroxy-3-methylglutarylcoenzyme a reductase and acylcoenzyme a: Cholesterol acyltransferase activities by phoshorylation-dephosphorylation in rabbit intestine

The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A: cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA: cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and ‘reactivation’ of enzyme activity. Cytosol caused a 78% increase in acyl-CoA: cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl₂ and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A: cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA: cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500×g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl₂, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA: cholesterol acyltransferase activity is observed.     

Coconut oil induces short-term changes in lipid composition and enzyme activity of chick hepatic mitochondria

We studied the short-term effects of a 20% coconut oil supplementation to the chick diet on lipid composition of liver and hepatic mitochondria, and changes that occurred in mitochondrial-associated enzymes as a result of this diet. No significant differences were observed in the lipid contents of liver when young chicks were fed the experimental diet, whereas hepatic mitochondria rapidly changed in response to this diet. Total cholesterol significantly increased in mitochondria at 24 hours of coconut oil diet feeding and decreased when dietary treatment was prolonged for 5 to 14 days. Changes in total mitochondrial phospholipids showed an inverse profile. A significant decrease in phosphatidylethanolamine and an increase in sphingomyelin were found at 24 hours. The cholesterol/phospholipid molar ratio significantly and rapidly (24 hours) increased in mitochondria from treated animals. Cytochrome oxidase activity drastically increased after 24 hours of experimental diet feeding and lowered to the control values when dietary manipulation was prolonged for 5 to 14 days. ATPase activity showed an inverse profile. Changes in cytochrome oxidase activity were parallel to changes in the cholesterol/phospholipid molar ratio, whereas changes in ATPase activity showed an inverse correlation with changes in this molar ratio. To our knowledge, this is one of the first reports on the very rapid response (24 hours) of mitochondrial lipid composition and function to saturated fat feeding.     

Hydroxymethylglutaryl CoA reductase and the modulation of microsomal cholesterol content by the nonspecific lipid transfer protein

The influence of membrane cholesterol content on 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase, EC 1.1.1.34) in rat liver microsomes was investigated. Microsomes were enriched in cholesterol by incubation with egg phosphatidylcholine-cholesterol vesicles and the nonspecific lipid transfer protein from rat liver. By this method, the microsomal cholesterol content was 2.5-fold enhanced up to final concentrations of 140 nmol cholesterol per mg microsomal protein. In another experiment, microsomes isolated from rats fed a cholesterol-rich diet were depleted of cholesterol by incubation with egg phosphatidylcholine vesicles and the transfer protein. Both cholesterol enrichment and depletion had virtually no effect on the microsomal HMG-CoA reductase activity. In another set of experiments, normal rat liver microsomes were incubated with human serum, resulting in a rise of microsomal cholesterol content. This was reflected in an increase of acyl-CoA:cholesterol acyltransferase activity but failed to have an effect on HMG-CoA reductase.