Hepatic metabolism and secretion of a cholesterol-enriched lipoprotein fraction

A potentially important source of cholesterol secreted in bile is cholesterol-rich lipoproteins. However, the fate of the cholesterol carried in these lipoproteins after hepatic uptake has not been investigated. We harvested an apoE- and cholesterol-rich lipoprotein fraction (d 1.02-1.06 g/ml) from hypercholesterolemic rats and examined the acute effects of these lipoproteins on hepatic cholesterol metabolism, very low density lipoprotein (VLDL) secretion, and biliary lipid secretion. Administration of a lipoprotein bolus (20 mg of cholesterol) to rats resulted in a significant decrease in 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and a significant increase in acyl-coenzyme A:cholesterol acyltransferase activity over controls at 1 hr. Hepatic cholesteryl ester content increased 400% with no change in hepatic free cholesterol content or biliary cholesterol secretion. These cholesterol-rich lipoproteins delivered in the isolated perfused liver effected a fivefold increase in hepatic VLDL secretion with no change in composition. Therefore, cholesterol-rich lipoproteins do not acutely alter biliary cholesterol secretion. Rather, the majority of the cholesterol delivered to the liver in these lipoproteins is either esterified and stored as cholesteryl ester or resecreted as free and esterified cholesterol in hepatic VLDL.     

Sodium benzoate inhibits fatty acid oxidation in rat liver: effect on ammonia levels

Sodium benzoate inhibited PC and octanoic acid-mediated State 3 respiration rates by 39 and 29%, respectively, at 0.5 mM in isolated rat liver mitochondria. At 2 mM, benzoate did not affect State 3 respiration rates with either succinate or malate plus glutamate, indicating that it did not act as an uncoupler. The oxidation of palmitate and octanoate was inhibited by 39 and 54% at 2 mM benzoate in liver homogenates. Benzoate, at 10 mmol/kg caused significant decreases in the levels of hepatic ATP, CoA, and acetyl-CoA. Administration of sodium benzoate to rats caused a dose-dependent increase in hepatic ammonia levels. However, the inhibitory effect of benzoate on fatty acid oxidation is not mediated through ammonia since ammonium chloride, at 1 mM, did not inhibit PC or octanoate oxidation in mitochondria or their oxidation in liver homogenate. Our results warrant a reevaluation of the use of sodium benzoate in the treatment of hyperammonemia.     

Plasma and urinary levels of carnitine in different experimental models of hyperammonemia and the effect of sodium benzoate treatment

The effect of hyperammonemia on plasma and urinary levels of carnitine was studied in different groups of +/Y (normal) and spf/Y (chronically hyperammonemic) mice. Experimental models of acute and subacute hyperammonemia were prepared in +/Y and spf/Y mice by the use of ammonium acetate ip injections and arginine-free diets, respectively. In acute hyperammonemia, the plasma levels of both free and acylcarnitines increased significantly whereas acyl/free carnitine ratio was decreased, indicating a mobilization of carnitine from the storage sites. The subacute hyperammonemia model showed the same tendency in respect of plasma and urinary carnitines; however, the values in plasma were more significantly different. The effect of sodium benzoate on plasma carnitine levels, during both an acute and a prolonged treatment, consisted in a significant lowering of free carnitine and a significant increase in the acyl/free carnitine ratio, in both +/Y normal and spf/Y mouse models. The changes in the urinary profile, on benzoate treatments, were not significant. These results demonstrate the individual effects of hyperammonemia and benzoate therapy on carnitine metabolism, which may be helpful in understanding and ameliorating the therapeutic approach to hereditary hyperammonemias.     

Effects of treatment with clofibrate, bezafibrate, and ciprofibrate on the metabolism of cholesterol in rat liver microsomes

The effects of treatment of rats with clofibrate, bezafibrate, and ciprofibrate on the hepatic metabolism of cholesterol were studied in rat liver microsomes. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase activity, regulating cholesterol biosynthesis, was unaffected by clofibrate and ciprofibrate and slightly decreased (20%) by bezafibrate. Also cholesterol 7 alpha-hydroxylase activity, governing bile acid biosynthesis, was unaffected by clofibrate and was reduced by 25-30% in the two other groups of rats. A major new finding was that all three fibric acid derivatives reduced ACAT (acyl-coenzyme A:cholesterol acyltransferase) activity, catalyzing the esterification of cholesterol, by 50-70%. The hepatic content of free and esterified cholesterol was determined in the bezafibrate-treated rats. The concentration of microsomal cholesteryl ester was about 60% lower in the treated rats compared to the controls whereas the concentration of total cholesterol was unchanged.     

The potentiation of ammonia toxicity by sodium benzoate is prevented by L-carnitine

Sodium benzoate has been recommended and even been used for the treatment of hyperammonemia in humans. More recently, a note of caution was raised since it has been shown that in experimental animals, sodium benzoate potentiates ammonia toxicity and inhibits urea synthesis in vitro. This has been further confirmed in the work presented here and the mechanism by which benzoate increases mortality and the levels of blood ammonia in mice given ammonium acetate have also been studied. In hyperammonemia, urea production and N-acetylglutamate levels were decreased by sodium benzoate. Pretreatment of mice with L-carnitine suppressed mortality following ammonium acetate plus sodium benzoate administration. Under these conditions L-carnitine lowered blood ammonia and increased urea production and N-acetylglutamate levels.     

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.     

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.     

Changes in cephalic and peripheral use of glucose and glutamine under the influence of hyperammonemia in rats]

Arterio-venous differences of glucose and glutamine were determined across the brain and across the hind limb in normal and ammonium salt infused rats, before and during an insulin tolerance test, in an attempt to study the effect of hyperammonemia on cephalic and muscular metabolism. The results demonstrate that 1) hyperammonemia reduces the hind limb uptake of glucose without affect the cephalic uptake of glucose which is lowered during hypoglycemia, 2) the reduction of the cephalic and muscular glutamine output induced by the hypoglycemia is masked in presence of an hyperammonemia. In conclusion, it may be assume that, at the concentration obtained in this study, hyperammonemia does not act directly in the pathogenesis of hepatic coma in which a decrease in cerebral glucose uptake described; on the other hand, ammonium plays an important role in the muscle metabolism.     

Mitochondrial urea cycle enzymes in rats treated with sodium benzoate

Since sodium benzoate, which is widely used to treat hyperammonemia its effect on mitochondrial urea cycle enzymes was investigated. its effect on mitochondrial urea cycle enzymes was investigated. Sodium benzoate was administered to urease treated hyperammonemic rats and controls. In both groups no interference with the activity of carbamylphosphate synthetase, ornithine carbamyltransferase and N-acetylglutamate synthetase in the liver could be observed at concentrations of benzoate in plasma found in hyperammonemic patients. Careful monitoring of plasma levels reduces benzoate toxicity as shown in a patient with argininosuccinic aciduria.     

A single therapeutic dose of valproate affects liver carbohydrate, fat, adenylate, amino acid, coenzyme A, and carnitine metabolism in infant mice: possible clinical significance

We have previously reported that chronic valproate administration reduced ketonemia in suckling mice and fasting epileptic children. The present study demonstrates that even a single dose of valproate in the therapeutic range for man caused a prolonged reduction of plasma beta-hydroxybutyrate levels in normal infant mice; the plasma glucose concentration was also significantly lowered. In the livers of these animals, there were extraordinary decreases in levels of free coenzyme A, acetyl CoA and free carnitine. Concomitantly concentrations of acid-soluble fatty acid (short-chain, non-acetyl) coenzyme A esters and of acid-insoluble (long-chain) fatty acid carnitine esters increased. There was evidence for inhibition of the metabolic flux through the Krebs citric acid cycle at those enzyme reactions which require coenzyme A. While valproate doubled liver alanine levels, concentrations of liver aspartate, glutamate and glutamine were reduced. All of the valproate-induced metabolite changes can be explained by the decrease of coenzyme A due to the accumulation of acid-soluble (non-acetyl) coenzyme A esters (presumably valproyl CoA and further metabolites). Decreased coenzyme A would limit the activities of one or more enzymes in the pathway of fatty acid oxidation and the Krebs citric acid cycle. Secondary decreases in acetyl CoA would limit both ketogenesis and gluconeogenesis. Decreased levels of selected hepatic amino acids could reflect their use as alternative fuels. The effect of clinical doses of valproate in infant mice may relate to the valproate-associated syndrome of hepatic failure and Reye-like encephalopathy in some infants and children and suggest a simple screen for those who may be at particular risk.     

Brain uptake of tryptophan in urease-injected hyperammonemic rats after treatment with benzoate or hippurate

The tryptophan uptake into forebrain and brainstem was measured in urease-injected rats and in controls after prior application of sodium benzoate or hippurate. Benzoate led to an increased brain uptake index of tryptophan. This effect was additive to the increase in tryptophan uptake effected by the provoked hyperammonemia. In contrast, hippurate did not alter tryptophan uptake across the blood-brain barrier. We conclude that benzoate should be monitored especially when applied intravenously to patients with hyperammonemic coma.     

Overexpression of SR-BI in hamsters treated with a novel ACAT inhibitor (F12511)

The effect of a novel acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor on cholesterol metabolism was studied in hamsters. Oral administration of F12511 (10 mg/kg/d) for 4 weeks produced a decrease in dietary cholesterol absorption (-18%) and in the liver concentration of esterified cholesterol (-75%), as compared with control values in untreated hamsters. While the hepatic expression of LDLr was unchanged by the treatment, that of SR-BI was increased (+142%), which suggests that the hepatic expression of SR-BI could be upregulated by a depletion of the cholesterol stores, due to ACAT inhibition. This SR-BI overexpression, however, did not induce a fall in plasma HDL-cholesterol concentration, in contrast with previous reports in transgenic mice overexpressing SR-BI at a higher extent.     

Influence of the acyl-CoA: cholesterol O-acyltransferase inhibitor, CL 277082, on cholesteryl ester accumulation in rabbit macrophage-rich granulomas and hepatic tissue

The influence of the acyl-CoA: cholesterol O-acyltransferase (ACAT) inhibitor, CL 277082, on macrophage cholesteryl ester accumulation in a rabbit carrageenan granuloma macrophage-foam cell model was studied. Diets were supplemented with 0.3% cholesterol and 6% peanut oil with or without the inhibitor (0.25%) for 4 weeks prior to granuloma induction, and macrophage-rich granuloma tissue was harvested 14 days after carrageenan injection. Serum cholesterol was monitored biweekly, and plasma lipoproteins were isolated terminally. Total, free and esterified cholesterol contents were measured in hepatic and granuloma tissue. In hepatic tissue, administration of CL 277082 resulted in an 80% reduction in the content of total cholesterol, a 37% decrease in free cholesterol, and a 90% decrease in esterified cholesterol. Similarly, in macrophage-rich granuloma tissue, total cholesterol content was decreased by 44%, and esterified cholesterol content by 61%, with no change in free cholesterol. Additionally, CL 277082 was shown to inhibit granuloma tissue ACAT activity by 45%, VLDL mass was decreased slightly, LDL mass increased 3.4-fold and HDL mass was similar in both the inhibitor-treated and control animals. CL 277082 resulted in a 57% decrease in VLDL cholesteryl ester content and a 4.5-fold increase in triacylglycerol. Cholesteryl ester content in LDL was decreased by 31% and LDL triacylglycerol was increased 5.2-fold, while the only change in HDL composition was a 3.5-fold increase in triacylglycerol. The reductions in both hepatic tissue and macrophage-rich granuloma tissue esterified cholesterol accumulation are considered to be due largely to cellular ACAT inhibition, and the altered distribution and composition of the plasma lipoproteins.     

Rat carboxylesterase ES-4 enzyme functions as a major hepatic neutral cholesteryl ester hydrolase

Although esterification of free cholesterol to cholesteryl ester in the liver is known to be catalyzed by the enzyme acyl-coenzyme A:cholesterol acyltransferase, ACAT, the neutral cholesteryl ester hydrolase (nCEH) that catalyzes the reverse reaction has remained elusive. Because cholesterol undergoes continuous cycling between free and esterified forms, the steady-state concentrations in the liver of the two species and their metabolic availability for pathways, such as lipoprotein assembly and bile acid synthesis, depend upon nCEH activity. On the basis of the general characteristics of the family of rat carboxylesterases, we hypothesized that one member, ES-4, was a promising candidate as a hepatic nCEH. Using under- and overexpression approaches, we provide multiple lines of evidence that establish ES-4 as a bona fide endogenous nCEH that can account for the majority of cholesteryl ester hydrolysis in transformed rat hepatic cells and primary rat hepatocytes.     

Chronic hyperammonemia in vivo impairs long-term potentiation in hippocampus by altering activation of cyclic GMP-dependent-protein kinase and of phosphodiesterase 5

Long-term potentiation (LTP) is impaired in the CA1 area of hippocampal slices from rats with chronic moderate hyperammonemia. We studied the mechanisms by which hyperammonemia in vivo impairs LTP. This process requires sequential activation of soluble guanylate cyclase, cyclic GMP-dependent protein kinase (PKG) and cyclic GMP-degrading phosphodiesterase. Application of the tetanus induced a rapid increase of cyclic GMP in slices from control or hyperammonemic rats, which is followed in control slices by a sustained decrease in cyclic GMP due to sustained activation of cyclic GMP-degrading phosphodiesterase, which in turn is due to sustained activation of PKG. In slices from rats with chronic hyperammonemia tetanus-induced decrease in cyclic GMP was delayed and transient due to lower and transient activation of PKG and of the phosphodiesterase. Hyperammonemia-induced impairment of LTP may be involved in the alterations of cognitive function in patients with hepatic encephalopathy.     

Biosynthesis of estradiol-17 beta fatty acyl esters by microsomes derived from bovine liver and adrenals

A fatty acyl coenzyme A:estradiol-17 beta acyl transferase activity has been detected in bovine hepatic and adrenocortical microsomes. It is thoroughly increased when adenosine triphosphate (5 mM) and coenzyme A (1 mM) are added to incubation buffer. Using a substrate concentration of 185 microM, the hepatic and adrenocortical microsomal activities have been found to be to 2.4 +/- 0.1 and 5.5 +/- 0.2 nmol/h/mg prot., respectively. Five major estradiol-17-esters have been isolated by reverse phase high performance liquid chromatography from both microsomal incubations, the fatty acid moieties being: arachidonate, linoleate, oleate, palmitate and stearate. However, the distribution of hepatic metabolites is quite different from that obtained with adrenocortical membranes, this is well explained by the corresponding differences between the endogenous contents of free fatty acids. With any of the two types of microsomal membranes used, the results show that estradiol is more susceptible to be esterified to polyunsaturated fatty acids than saturated ones. The possible physiological implications of such an activity in liver and adrenals are discussed.     

Hepatic origin of hyperammonemia induced by shock in normal rats.

This study was undertaken in order to see whether instability in blood ammonia levels frequently observed in the anaesthesized rat could be explained by variations in blood pressure. In normal Wistar rats, anaesthesized with sodium pentobarbital, a bleeding of 1 ml/100 g body weigth produced in a few minutes a significant decrease in blood pressure and a significant increase in arterial blood ammonia level. With the blood pressure normalization following the blood reinfusion this hyperammonemia decreased but reappeared at high levels after a second and a third bleeding. The shock produced by an occlusion of the inferior vena cava above the renal veins gave similar results. The arterial hyperammonemia induced by shock did not result from muscle or renal ammonia production but is related to impaired hepatic uptake of portal ammonia. These results indicate that in the rat the blood pressure stability is a necessary precondition in any experiment on ammonia metabolism.     

Evaluation of kidney and liver subacute toxicity induced by Bezalip-Pravastatin-Lopid antihyperlipidaemic compounds in rats

Renal and hepatic subacute toxicity induced by the antihyperlipidaemic drugs: Bezalip-Pravastatin and Lopid was investigated in rats using serum biochemical parameters. Toxicological evaluation was performed in serum samples following the administration of the therapeutic dose regimens of the compounds that were previously shown to be effective in inhibition of 3-hydroxy-methylglutaryl coenzyme A (HMG CoA) reductase, the enzyme controlling the rate-limiting step in the synthesis of cholesterol, and acyl-CoA cholesterol acyl transferase (ACAT) which converts intracellular free cholesterol to cholesterol ester. Renal and hepatic subacute toxicity was evaluated by measuring enzyme activity or concentrations of: alanine aminotransferace, alkaline phosphatase, aspartate aminotransferase, gamma-glutamyltransferase, glucose, potassium, sodium, blood urea nitrogen, uric acid and creatinine. The use of the above serum biochemical parameters indicated that the overall toxicity impact of antihyperlipidaemic drugs was Bezalip = Pravastatin < Lopid. We have found that the Pravastatin--in contrast to the above antihyperlipidaemic drugs--only transiently affects the biochemical parameters associated with toxicity, but, it affects some of the biochemical parameters associated with hepatic and renal toxicity, up to a significantly lower extent than the antihyperlipidaemic drugs.     

Effect of progesterone on rat plasma and liver lipid levels (author's transl)

The effect of progesterone upon several lipidic parameters on rat liver and plasma was studied. Progesterone administration led to a significant increase in the hepatic triacylglyceride and cholesterol esterified levels and to a decrease in the phospholipid content. After progesterone treatment decreases in plasma total lipids, phospholipids, cholesterol and cholesterol esterified were observed, whereas plasma triacylglyceride and free fatty acid levels increased. The hormonal treatment altered the lipoprotein pattern, which significantly reduced the beta-lipoprotein fraction.     

Effect of 17 alpha-ethinylestradiol on the activity of enzymes synthesizing and hydrolyzing cholesterol esters in the rat liver

Intraperitoneal injection of 25 micrograms/100 g body weight of 17 alpha-ethinyl estradiol to rats was shown to decrease serum cholesterol and to increase hepatic cholesterol. The rise in the level of non-labeled and C14-labeled free and esterified cholesterol in hepatic homogenate, as well as in lysosomal and cytosol fractions was accompanied by reduced activity of acyl-CoA-cholesterol acyltransferase and increased activity of lysosomal cholesterol esterase, as compared with the controls. The activity of cytoplasmic cholesterol esterase remained practically unchanged. Fistula bile of treated rats collected during 30 min was analyzed for the concentration of free cholesterol and bile acids. It has been shown that treatment of rats with 17 alpha-ethinyl estradiol caused an increase in hepatic cholesterol elimination via bile pathways.