Acyl-CoA synthetase and the peroxisomal enzymes of beta-oxidation in human liver. Quantitative analysis of their subcellular localization.

The presence of acyl-CoA synthetase (EC 6.2.1.3) in peroxisomes and the subcellular distribution of beta-oxidation enzymes in human liver were investigated by using a single-step fractionation method of whole liver homogenates in metrizamide continuous density gradients and a novel procedure of computer analysis of results. Peroxisomes were found to contain 16% of the liver palmitoyl-CoA synthetase activity, and 21% and 60% of the enzyme activity was localized in mitochondria and microsomal fractions respectively. Fatty acyl-CoA oxidase was localized exclusively in peroxisomes, confirming previous results. Human liver peroxisomes were found to contribute 13%, 17% and 11% of the liver activities of crotonase, beta-hydroxyacyl-CoA dehydrogenase and thiolase respectively. The absolute activities found in peroxisomes for the enzymes investigated suggest that in human liver fatty acyl-CoA oxidase is the rate-limiting enzyme of the peroxisomal beta-oxidation pathway, when palmitic acid is the substrate.

The effect of progesterone on the regulatory mechanisms of biliary cholesterol secretion in the rat.

We tested the hypothesis that progesterone, an inhibitor of cholesterol esterification in liver microsomes, increases biliary cholesterol output by increasing the availability of cholesterol. Initial bile samples of 20 min were obtained from acute bile fistula rats after seven daily doses of progesterone (5 to 55 mg per kg of body weight). Biliary cholesterol output correlated with the doses of progesterone, r = 0.64 (p less than 0.005). A 100% increment in biliary cholesterol output was obtained with progesterone doses of 30 to 55 mg per kg of body weight. Under these conditions, biliary phospholipid output increased 50% (p less than 0.02), but bile salt output remained normal. The relationship between biliary cholesterol and phospholipids as a function of bile salt output was studied after acute depletion of the bile salt pool. A rectangular hyperbola was the best curve fitting for the experimental data in control and progesterone-injected rats. In the physiological range of bile salt output, between 60 and 120 nmoles per gm per min, progesterone-injected rats secreted 100% more biliary cholesterol than did controls. The calculated theoretical maximal cholesterol and phospholipid outputs were significantly increased in progesterone-injected animals. Serum and hepatic cholesterol pool, free and ester fractions, remained normal. The acyl-CoA: cholesterol acyltransferase reaction was 30% inhibited in hepatic microsomes of progesterone-injected rats, (p less than 0.05). The changes in biliary phospholipids and cholesterol output produced by progesterone were rapidly reversed by either 0.5% cholesterol feeding or 2 mg per kg of body weight ethynyl estradiol injection. These manipulations simultaneously produced a 100% increment in the microsomal acyl-CoA: cholesterol acyltransferase activity (p less than 0.005) and increased 4-fold the concentration of hepatic cholesterol esters. This experimental model suggests a functional interrelationship between biliary cholesterol output and the rate at which the liver esterifies cholesterol.

Hepatic cholesterogenesis in Chileans with cholesterol gallstone disease. Evidence for sex differences in the regulation of hepatic cholesterol metabolism.

To test the hypothesis that hepatic cholesterol synthesis and concentration are specifically increased in women and men with cholesterol gallstone disease, we studied in a series of 24 surgical liver biopsy specimens, the rate of acetate incorporation into cholesterol and CO2, and the concentration of free and esterified cholesterol. There were 6 women in each group of 12 control and 12 gallstone patients. Therefore, hypersecretion of biliary cholesterol, a common finding in Chileans with gallstones, is not pathologically linked to a basic biochemical abnormality in the hepatic cholesterogenic pathway. The concentrations of hepatic cholesterol were similar in patients with and without gallstones. However, there were significant increments of 37% in the free fraction and 78% in the esterified fraction of hepatic cholesterol in female subjects, irrespectively of the presence of cholelithiasis. These results suggest that the flux of lipoprotein cholesterol to the liver is increased in women. The regulatory mechanism of hepatic cholesterogenesis is less sensitive to cellular cholesterol in women than in men, as the fluxes of acetate into cholesterol are similar to both sexes, in spite of the significant increase of hepatic cholesterol in females. These findings demonstrate that sex factors play a fundamental role in the regulation of cholesterol metabolism in the human liver.

Bile acid pool changes and regulation of cholate synthesis in experimental diabetes.

The effect of alloxan-diabetes and insulin treatment in bile acid pool size and composition, bile acid secretion and cholic acid synthesis was investigated in the rat. The size of the cholate pool was significantly increased 4 days after diabetes induction. It reached a constant size three times that of control animals after 2 weeks of diabetes. Changes in bile acid pool size and secretion were directly dependent of the insulin deficiency state since they were reversed by insulin treatment and were not influenced by the caloric intake of the animal nor the pharmacologic effect of alloxan. Biliary cholate secretion was also 3-fold increased in diabetic rats and it accounted for more than 80% of the total bile acids compared to 60% in the control group. The calculated daily rate of cholate synthesis was increased in diabetic rats and the circadian rhythm of cholate synthesis was abolished in this condition. Therefore, it was shown that the negative feedback mechanism that regulates bile acid snythesis was deleted in diabetes. This mechanism was partially restored after 2 weeks of insulin treatment. These studies demonstrated that bile acid metabolism was profoundly changed in alloxan-diabetic rats and suggested that insulin may play an important role in the regulation of bile acid snythesis and intestinal absorption.

The mechanisms of and the interrelationship between bile acid and chylomicron-mediated regulation of hepatic cholesterol synthesis in the liver of the rat.

Hepatic cholesterol synthesis is controlled by both the size of the bile acid pool in the enterohepatic circulation and by the amount of cholesterol reaching the liver carried in chylomicron remnants. These studies were undertaken to examine how these two control mechanisms are interrelated. When the size of the pool was systematically varied, the logarithm of the rate of hepatic cholesterol synthesis varied in an inverse linear fashion with the size of the taurocholate pool between the limits of 0 and 60 mg of bile acid per 100 g of body weight. The slope of this relationship gave the fractional inhibition of cholesterol synthesis associated with expansion of the taurocholate pool and was critically dependent upon the amount of cholesterol available for absorption from the gastrointestinal tract. Furthermore, the degree of inhibition of cholesterol synthesis in the liver seen with taurocholate feeding was reduced by partially blocking cholesterol absorption with beta-sitosterol even though the bile acid pool was still markedly expanded. In rats with diversion of the intestinal lymph from the blood, a five-fold expansion of the taurocholate pool resulted in only slight suppression of the rate of hepatic cholesterol synthesis, and even this inhibition was shown to be attributable to small amounts of cholesterol absorbed through collateral lymphatic vessels and (or) to a fasting effect. Similarly, the infusion of either taurocholate or a combination of taurocholate and taurochenate into rats with no biliary or dietary cholesterol available for absorption caused no suppression of hepatic cholesterol synthesis. Finally, the effect of changes in the rate of bile acid snythesis on hepatic cholesterol synthesis was examined. The fractional inhibition of cholesterol synthesis found after administration of an amount of cholesterol sufficient to raise the hepatic cholesterol ester content by 1 mg/g equalled only --0.36 when bile acid snythesis was increased by biliary diversion but was --0.92 when bile acid synthesis was suppressed by bile acid feeding. It is concluded that (a) bile acids are not direct effectors of the rate of hepatic cholesterol synthesis, (b) most of the inhibitory activity seen with bile acid feeding is mediated through increased cholesterol absorption, and (c) bile acids do have an intrahepatic effect in that they regulate hepatic cholesterol synthesis indirectly by altering the flow of cellular cholesterol to bile acids.

Rate constants for the uptake of cholesterol from various intestinal and serum lipoprotein fractions by the liver of the rat in vivo.

The increase in the mass of cholesterol esters in the liver was used to estimate hepatic net uptake rates of cholesterol from various serum and intestinal lipoprotein fractions. Initial uptake rates equalled essentially zero for high density serum lipoproteins and for large chylomicrons while administration of both low density serum lipoproteins and smaller chylomicrons produced a significant increase in hepatic cholesterol ester levels. The rate of uptake of both serum lipoprotein fractions did not change over a 5 h interval after injection: in contrast, the rates of uptake of the intestinal fractions increased 10-25-fold during this interval. Circulation of large chylomicrons in functionally eviscerated rats markedly increased the rate of hepatic cholesterol uptake when these metabolized lipoproteins were reinjected into recipient animals. Uptake of cholesterol from intestinal lipoproteins was essentially a linear function of the amount of chylomicrons administered to the animals and was independent of the level of circulating serum cholesterol and the rate of hepatic cholesterogenesis. These observations are consistent with the view that the liver is capable of taking up cholesterol from chylomicron remnants and, at significantly lower rate, low density serum lipoproteins.

Dissociation of beta-hydroxy-beta-methylglutaryl-CoA reductase activity from the overall rate of cholesterol synthesis in the liver following the intravenous administration of lipid.

After the intravenous administration to the intact rat of triglyceride carried in either intestinal lipoproteins or in an artificial fat emulsion, the enzymatic activities of microsomal beta-hydroxy-beta-methylglutaryl-CoA reductase activity in the liver assayed in vitro became markedly elevated. This elevation of enzyme activity was not associated with a corresponding change in the overall rate of cholesterol synthesis in the rat liver slice as measured by the incorporation of either [3H]water or [1-14C]octanoate into nonsaponifiable lipids or into digitonin-precipitable sterols. The degree of dissociation of hydroxymethylglutaryl-CoA reductase activity from the overall rate of cholesterol synthesis correlated closely with the amount of lipid administered to the animal, the level of circulating lipids, and the level of ketone synthesis manifest in the liver cell suggesting that this phenomenon might be the consequence of a detergent effect of elevated cellular levels of fatty acids. In any event, under these experimental circumstances hydroxymethylglutaryl-CoA redutase activity no longer reflects the rate at which the liver cell is synthesizing cholesterol.

Ability of six different lipoprotein fractions to regulate the rate of hepatic cholesterogenesis in vivo.

Two in vivo assay procedures were used to study the inhibitory activity of cholesterol carried in three intestinal lymph and three serum lipoprotein fractions on the rate of cholesterol synthesis in the liver. In the first preparation, different lipoproteins were injected intravenously as a bolus into rats at the mid-light phase of the diurnal light cycle, following which they were killed 12 hours later in the mid-dark phase of the cycle. Using this assay, three intestinal lymph lipoprotein fractions of varying Sf values all produced a similar degree of inhibition which averaged approximately 11%/mg of cholesterol injected. The serum lipoprotein fractions caused only about one-third this amount of inhibition. Detailed analysis of events occurring within the liver during this 12-hour assay period revealed that there were marked differences in the rate of net cholesterol uptake into the liver and in the rate of new removal of cholesterol esters from the liver following injection of each of these different lipoprotein fractions. The amount of inhibition of sterol synthesis produced by any fraction was proportional to the product of the incremental increase in hepatic cholesterol ester content and the time over which this increase in esters occurred. In the second type of assay where the lipoprotein fractions were administered to the animals as a continuous intravenous infusion over 24 hours the largest increase in hepatic cholesterol ester content and the greatest inhibition of cholesterol synthesis was found with intestinal lipoproteins having Sf values larger than 8000. Intestinal lipoprotein fractions with lower Sf values and all serum lipoprotein fractions were significantly less effective in bringing about an increase in hepatic cholesterol ester content and in producing inhibition of cholesterol synthesis by the liver. These studies emphasize the primary role of cholesterol carried in lipoproteins of intestinal origin in regulating hepatic sterol synthesis. The inhibitory activity of these fractions appears to correlate with the ability of these lipoproteins to bring about a maximal increase in hepatic cholesterol ester content which, in turn, appears to relate to the capacity of these fractions to transfer cholesterol rapidly into the hepatocyte while, at the same time, slowing the rate of cholesterol mobilization from the liver.

The kinetic characteristics of inhibition of hepatic cholesterogenesis by lipoproteins of intestinal origin.

In these studies intestinal lipoproteins were injected intravenously into recipient rats in order to study the kinetic characteristics of cholesterol uptake by the liver cell and inhibition of the cholesterol synthetic pathway. Net cholesterol uptake from circulating intestinal lipoproteins took place only in the liver, and only this tissue manifested inhibition of cholesterol syntheses. Cholesterol uptake by the liver, quantified by a rise in the cholesterol ester content, was a linear function of time and of the amount of lipoprotein cholesterol administered to the animals. Using groups of rats that were either fed cholesterol or injected intravenously with intestinal lipoproteins as a bolus or as a continuous infusion, there was generally a correlation between inhibition of the rate of cholesterol synthesis and the cholesterol ester content of the liver. However, there was no consistent quantitative relationship between these two variables suggesting either that cholesterol ester was not the immediate effector of the inhibition or, alternatively, that there was intracellular localization of the effector at the site of control of the rate-limiting enzyme in the cholesterogenic pathway.