Pancreatic cholesterol esterases. 3. Kinetic characterization of cholesterol ester resynthesis by the pancreatic cholesterol esterases

The ability of cholesterol esterase to catalyze the synthesis of cholesterol esters has been considered to be of limited physiological significance because of its bile salt requirements for activity, though detailed kinetic studies have not been reported. This study was performed to determine the taurocholate, pH, and substrate requirements for optimal cholesterol ester synthesis catalyzed by various pancreatic lipolytic enzymes, including the bovine 67- and 72-kDa cholesterol esterases, human 100-kDa cholesterol esterase, and human 52-kDa triglyceride lipase. In contrast to current beliefs, cholesterol esterase exhibits a bile salt independent as well as a bile salt dependent synthetic pathway. For the bovine pancreatic 67- and 72-kDa cholesterol esterases, the bile salt independent pathway is optimal at pH 6.0-6.5 and is stimulated by micromolar concentrations of taurocholate. For the bile salt dependent synthetic reaction for the 67-kDa enzyme, increasing the taurocholate concentration from 0 to 1.0 mM results in a progressive shift in the pH optimum from pH 6.0-6.5 to pH 4.5 or lower. In contrast, cholesterol ester hydrolysis by the 67-, 72-, and 100-kDa enzymes was characterized by pH optima from 5.5 to 6.5 at all taurocholate concentrations. Optimum hydrolytic activity for these three enzyme forms occurred with 10 mM taurocholate. Since hydrolysis is minimal at low taurocholate concentrations, the rate of synthesis actually exceeds hydrolysis when the taurocholate concentration is less than 1.0 mM. The 52-kDa enzyme exhibits very low cholesterol ester synthetic and hydrolytic activities, and for this enzyme both activities are bile salt independent. Thus, our data show that cholesterol esterase has both bile salt independent and bile salt dependent cholesterol ester synthetic activities and that it may catalyze the net synthesis of cholesterol esters under physiological conditions.     

Regulation of gallbladder cholesterol concentration in the hamster. Role of hepatic cholesterol level

The goal of this investigation was to determine how alterations in hepatic cholesterol metabolism influence the cholesterol content of gallbladder bile in hamsters. Although the rate of hepatic cholesterol synthesis was varied over 600-fold, there was no direct relationship between the rate of cholesterol synthesis and the cholesterol content of gallbladder bile. However, expansion of the hepatic cholesterol pool by 42-fold resulted in an 11-fold increase in gallbladder bile cholesterol. Examination of four subfractions of the hepatic cholesterol pool revealed that the cholesterol content of gallbladder bile was most consistently correlated with the free cholesterol level in both hepatic tissue and hepatic microsomes from all experimental groups. In most groups of animals in which gallbladder bile cholesterol was increased, plasma lipoprotein cholesterol levels were also increased. It was concluded that in hamsters, under these experimental conditions, changes in the cholesterol content of gallbladder bile were directly related to alterations in cholesterol content of the liver and most closely related to alterations in the free cholesterol content of that tissue.     

Influence of fasting and cholesterol feeding on the cholesterol synthesis in rats in vivo.

An inhibition of the cholesterol synthesis located between lanosterol and cholesterol could be shown in fasting rats in vivo with glucose, palmitate, acetate, and mevalonate as tracer substances. The same type of inhibition of the cholesterol synthesis was observed after cholesterol feeding. No indication was obtained for any other inhibiting effect located between acetyl-coenzyme A and lanosterol by these in vivo experiments.     

Apolipoproteins, membrane cholesterol domains, and the regulation of cholesterol efflux.

Published data related to both cell membrane biology and apolipoprotein structure are reviewed and used to formulate a new model describing the mechanisms of cholesterol efflux from cell plasma membrane to high density lipoprotein (HDL) particles. The central premise of this model is the existence of heterogenous domains of cholesterol within plasma membranes. We propose that cholesterol efflux from cell membranes is influenced by three factors: 1) the distribution of cholesterol between cholesterol-rich and cholesterol-poor membrane domains, 2) the diffusion of cholesterol molecules through the extracellular unstirred water layer, and 3) the transient interaction of segments of the amphipathic helix of the HDL apolipoprotein with cholesterol-poor membrane domains resulting in enhanced cholesterol efflux.     

Triglyceride-rich lipoprotein cholesterol exceeds low-density lipoprotein cholesterol in hypertriglyceridemia patients.

The atherogenicity of triglyceride-rich lipoprotein has been revealed. This study was performed to explore the clinical importance of triglyceride-rich lipoprotein by measuring its cholesterol content and comparing it with other lipoprotein fractions. Blood samples were obtained from 103 patients whose fasting plasma triglyceride concentration exceeded 300 mg/dl. The cholesterol monitor using the technique of high-performance liquid chromatography was used for the measurement of their plasma cholesterol concentrations and the determination of cholesterol distribution among lipoprotein fractions. This monitor showed 4 peaks: large-triglyceride-rich lipoprotein, small-triglyceride-rich lipoprotein, low-density lipoprotein, and high-density lipoprotein. Total cholesterol increased with increasing triglyceride. The increment of total cholesterol was nearly equal to that of small-triglyceride-rich lipoprotein cholesterol. Small-triglyceride-rich lipoprotein cholesterol exceeded low-density lipoprotein cholesterol where plasma triglyceride concentration was over 500 mg/dl. In conclusion, triglyceride-rich lipoprotein may be clinically important for hypertriglyceridemic patients as a source of cholesteryl ester in arteriosclerotic plaques, and increased triglyceride-rich lipoprotein cholesterol may be used as a basis for hypertriglyceridemia atherogenicity. Our study suggests that hypertriglyceridemia should be treated to prevent arteriosclerotic disease.     

Regulation of neutral cholesterol esterase and acyl-CoA : cholesterol acyltransferase in the rat adrenal gland.

The activities of neutral cholesterol esterase and acyl-CoA : cholesterol acyltransferase in rat adrenal gland were measured at various time intervals over 24 h. The activity of cholesterol esterase displayed diurnal rhythm, with a major peak at the onset of darkness coinciding with the peak in the diurnal rhythm of plasma corticosterone concentration. The activity of acyl-CoA : cholesterol acyltransferase also exhibited a characteristic diurnal rhythm, with the minimum activity occurring 3 h after the onset of darkness. The profile of the rhythm exhibited by the activity of the esterifying enzyme was similar to the mirror image of the pattern of diurnal rhythm in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase. Microsomal non-esterified cholesterol showed a gradual decline with a significant decrease in concentration at the onset of darkness, thus suggesting that diurnal removal of cholesterol in the environment of the esterifying enzyme and hydroxymethylglutaryl-CoA reductase leads to such diurnal decrease or increase in the activities of these two enzymes. Acute administration of corticotropin led to a 3-fold increase in the activity of cholesterol esterase, a 50% decrease in the activity of acyl-CoA : cholesterol acyltransferase and a 2-fold increase in the activity of hydroxymethylglutaryl-CoA reductase. Corticotropin administration also resulted in a significant decrease in microsomal non-esterified cholesterol and increase in plasma corticosterone concentration. These observations suggest that corticotropin plays an important part in generating the diurnal rhythm in the activities of the three enzymes.     

Lateral diffusion of cholesterol in monolayers.

The surface diffusion coefficient of cholesterol in cholesterol monolayers has been measured as a function of cholesterol surface concentration. Two different radiochemical methods, one integral and the other differential, were developed which gave comparable results. In the integral method two cholesterol monolayers, one of which is radioactive, are isolated on inert hydrophilic supports and then brought into contact. After some time the supports are separated and the radioactivity of the supports is measured. The differential method is an autoradiographic experiment. Two cholesterol monolayers, one of which is radioactive, are separated by means of a thin barrier. Upon removal of the barrier and at later times, an autoradiographic plate is brought to within a fraction of a mm from the aqueous surface and exposed. The plates are developed and analysed. The data show that the cholesterol surface diffusion coefficient in the dilute monolayers is approximately 10(-6)cm2/s and is nearly independent of surface concentration up to a concentration corresponding to an area of 40 A2/molecule. As the monolayer becomes compressed beyond this surface concentration, the diffusion coefficient decreases ubruptly with the deeply decreasing surface tension to about 10(-7) cm2/s, when a fully condensed surface layer of 38 A2/molecule is reached. This diffusion coefficient is of the same order of magnitude as the diffusion coefficients measured in lipid bilayers and in membranes.     

Microdetermination of cholesterol in serum lipoproteins.

A two-step procedure for the microdetermination of cholesterol in serum lipoproteins is compared with cholesterol quantitation after density gradient ultracentrifugation. Serum lipoproteins from 10 mul of serum are separated by electrophoresis on agarose and visualized by precipitation with dextran sulfate--CaCl2. The lipoprotein bands are cut off from the plates, the agarose slices are hydrolyzed by gas-liquid chromatography. The comparison between the two procedures reveals satisfactory correlations for beta-and pre-beta-lipoproteins and total serum. There is excellent recovery of cholesterol in fractionated lipoproteins.