Hepatic Cholesterogenesis Research Articles

Relationship between activity of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and secretion of very-low-density-lipoprotein cholesterol by the isolated perfused liver and in the intact rat

The hepatic output of triacylglycerol and cholesterol from very-low-density lipoprotein (VLD lipoprotein), and the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase were compared in the isolated perfused rat-liver preparation and in the intact rat. The output of triacylglycerol and cholesterol from VLD lipoprotein by the perfused liver was stimulated by oleate concomitant with stimulation of hepatic microsomal hydroxymethylglutaryl-coenzyme A reductase activity. In the intact animal treated with Triton WR-1339, the magnitude of secretion of triacylglycerol and cholesterol from VLD lipoprotein coincided with the diurnal rhythm of hepatic hydroxymethylglutaryl-coenzyme A reductase activity, which was maximal at 24:00 h and minimal at 12:00 h. These observations suggest that the stimulation of the reductase and of the secretion of cholesterol from VLD lipoprotein by non-esterified fatty acids, as observed with the isolated perfused rat liver preparation in vitro, may also be an important physiological mechanism in vivo. Hepatic cholesterogenesis may be stimulated under conditions conductive to the secretion of the VLD lipoprotein, the primary transport form for triacylglycerol in the postabsorptive state.

Colorimetric estimate of cholesterogenesis in livers perfused in the presence of trans-1,4-bis-(2-chlorobenzylaminomethyl)-cyclohexane dihydrochloride (AY-9944)

AY-9944 ¦ trans-1,4-bis-(2-chlorobenzylaminomethyl)-cyclohexane dihydrochloride¦ inhibits the conversion of sterol intermediates to cholesterol. The appearance of sterol intermediates, measured colorimetrically as “fast-acting” sterols, has been used as an estimate of hepatic cholesterogenesis in vivo after treatment with AY-9944. The appearance of fast-acting sterols as an estimate of cholesterogenesis was studied in the isolated rat liver, perfused in vitro in the presence of AY-9944, with or without oleic acid. Oleic acid elevated the appearance of fast-acting sterols in the liver and the perfusate. The secretion of fast-acting sterols by the perfused liver correlates with the activity ofmicrosomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of cholesterogenesis, and the synthesis of fast-acting sterols. The latter also correlates with the activity of HMG-CoA reductase. These observations are in agreement with previous reports of stimulation of the secretion and synthesis of cholesterol by liver perfused with oleic acid. Based on these findings, it is suggested that the level of fast-acting sterols after treatment with AY-9944 may be used as a colorimetric estimate for the synthesis and distribution of cholesterol in the isolated perfused rat liver.

The effect of cholestyramine on bile acid kinetics in healthy controls.

Serum lipids and the kinetics of the two primary bile acids, cholic acid (C) and chenodeoxycholic acid (CD), were studied in six normolipidaemic subjects before and during treatment with cholestyramine (12 g/day). After therapy, total serum and low-density lipoprotein cholesterol decreased by about 15%. These changes were accompanied by significant increases in the pool size, synthesis, and fractional turnover rate (FTR) of C and in the synthesis and FTR of CD. In spite of the enhanced formation of CD, the CD pool size decreased in all subjects, on average by more than 50%. The combined pool size of C and CD remained constant, but the mean total bile acid formation increased by a factor of 2.9. It is suggested that the different responses of C and CD, with an augmented contribution of C to the total amounts of bile acid produced, reflect an enhanced hepatic cholesterogenesis and, possibly, that C and CD to some extent originate from different cholesterol precursor pools.

Cholesterol synthesis in isolated rat hepatocytes. In vitro effect of sterols and bile acids

In this report we investigated the in vitro effects of various sterols and bile acids on the overall rate of cholesterol synthesis in isolated adult rat hepatocytes. When rat hepatocytes were incubated with Na-taurocholate or Na cholate (up to 50 μmol/l) no inhibition of cholesterogenesis was found. By contrast Na-glycochenodeoxycholate reduced cholesterogenesis to a significant extent, but only at concentrations (10 to 50 μmol/l) which were higher than those usually found in both the liver and the portal blood of the rat. When hepatocytes were incubated in the presence of protonated free bile acids (10 to 30 μmol/l) cholesterogenesis was greatly reduced. Mono and dihydroxy bile acids were more powerful inhibitors than the trihydroxy ones. The exposure of hepatocytes to free cholesterol (up to 200 μmol/l) produced only a slight reduction of cholesterogenesis (20%) whereas the addition of oxygenated sterols to the medium (7-ketocholesterol, 7-α-hydroxycholesterol and 25-hydrocholesterol) were found to inhibit cholesterogenesis at concentrations lower than 40 μmol/l. In conclusions, our findings raise the possibility that not cholesterol “per se” but rather some of its derivatives (both oxygenated sterols and free protonated bile acids) may be the modulators of hepatic cholesterogenesis.

Dietary Fat-Dependent changes in Hepatic Cholesterogenesis and the Activity of 3-Hydroxy-3-Methylglutaryl-CoA Reductase in Fasted-Refed Rats

Effects of various dietary fats on the activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and sterol and fatty acid synthesis from [1-14C]acetate and [2-14C]mevalonate were examined in the liver from fasted-refed rats. Rats fasted for 2 days were refed a fat-free diet or diets containing various fats (tricaprylin, trilaurin, trimyristin, tristearin, camellia oil, or safflower oil) at the 10% level for 1, 3, or 7 days. The activity of HMG-CoA reductase was restored to about one-half of the pre-fasting levels in all groups after refeeding for 1 day and increased to above the pre-fasting levels after 3 days, with the exception of safflower oil, the rise was especially noticeable when fat-free, tricaprylin, and tristearin diets were fed. After 7 days, the activity of HMG-CoA reductase, except for rats refed tristearin, was decreased to levels that were far below those observed after 1 day-refeeding. This was particularly marked with tricaprylin, trilaurin, and camellia oil. The response of sterogenesis resembled that of the reductase. Dietary fat-dependent modification of fatty acid synthesis from [1-14C]acetate was first demonstrated after 7 days. Hepatic esterified cholesterol tended to accumulate and the deposition was marked after 3 days of refeeding. However, fat-dependent alterations of this parameter were remarkable on day 7. The concentration of plasma cholesterol also showed dietary fat-dependent changes after refeeding. Dietary fats appear to play an important role not only in the regulation of hepatic HMG-CoA reductase and sterol synthesis, but also in the overall processes of cholesterol dynamics.

Cholesterol metabolism in the liver and intestine of the chick: Effect of dietary cholesterol, taurocholic acid and cholestyramine

The effect of feeding cholesterol, taurocholic acid, or cholestyramine to chicks on cholesterogenesis from [1-14C] acetate in liver and intestine was determined in vitro using tissue slices, and in vivo by i.v. injection of [14C] acetate. The conversion of cholesterol to bile acids in liver in vivo was measured in the same treatments after i.v. injection of [3H] cholesterol. Hepatic cholesterogenesis in vitro and in vivo was depressed by dietary cholesterol and taurocholate and enhanced by cholestyramine. Intestinal cholesterogenesis in vivo was depressed only by taurocholate whereas ileal cholesterogenesis in vitro was reduced by dietary cholesterol. Conversion of cholesterol to bile acids was enhanced by dietary cholesterol and cholestyramine and depressed by taurocholate. Hepatic cholesterol metabolism in the chick appears to be regulated by mechanisms similar to those reported for other species.

Effects of Exercise on Plasma and Liver Lipids of Rats

The effects of treadmill exercise on hepatic cholesterogenesis and fecal steroid excretion were studied using male Wistar rats fed a commercial pellet ration. Exercise, in comparison with ad libitum or pair-feeding sedentary groups, caused the following significant changes: (i) a reduction in the concentration of plasma triglyceride, phospholipid and cholesterol; (ii) a reduction in liver weight; (iii) increases in hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity and incorporation of mevalonate into digitonin precipitable sterol; (iv) increases in excretion of neutral and acidic steroids into the feces; (v) the increase in lipoprotein lipase activity in the cardiac muscle, and (vi) the decrease in the concentration of carcass triglyceride but not of cholesterol. These data suggest that the mechanism responsible for the plasma cholesterol-lowering effect of exercise is attributable to an increase in exretion of fecal neutral and acidic steroids accompanied by an acceleration of cholesterol turnover in the body.

Characterization of an Inhibitor of Hepatic Cholesterogenesis Present in Hepatic Microsomes

An inhibitor of hepatic cholesterol synthesis present in hepatic microsomes can be solubilized either by an acetone or an ethanol powder preparation. Other methods such as methanol and chloroform:methanol powder preparations and treatment with EDTA do not solubilize the factor. The factor appears to be proteinaceous since its activity is lost after exposure to proteolytic enzymes and heat treatment. In addition, the inhibitor does not require a phospholipid for activity. 3this inhibitor is stable for long periods (60 hrs.) at room temperature and can be isolated in good yield from liver maintained at 4 degrees C for 8 hours postmortem.

The sources of rat biliary cholesterol and bile acid.

The precursor sources of bile acid and bile neutral sterol were evaluated in the rat using Triparanol to inhibit the terminal reduction in the synthesis of cholesterol. During the initial period of Triparanol administration, the accumulation of hepatic desmosterol acts to segregate relatively newly synthetic hepatic sterol from the bulk of the equilibrated sterol mass. Biliary excretion of newly synthetic sterol can then be determined in acute studies, assuming no great differences between desmosterol and cholesterol as precursors of biliary neutral sterol or bile acid. It has been determined in this model that newly synthetic sterol comprises a mean of about 28% of the total biliary neutral sterol output. This fraction fell when hepatic cholesterogenesis was suppressed by prior cholesterol feeding. By using this approach in conjunction with the administration of labeled mevalonate to a renal pedicle-ligated rat, it was possible to calculate the amount of bile acid produced from either newly synthesized sterol or the equilibrated sterol pool. It has been estimated that the bulk of bile acid synthesis arises from this equilibrated source when these determinations were made within two hours of creating the fistula. With more prolonged fistula times, more of the bile acid originated from the newly synthesized sterol.

On the metchanism for the regulation of 3-hydroxy-3-methylglutaryl-coenzyme a reductase, of cholesterol 7α-hydroxylase and of acyl-coenzyme A: Cholesterol acyltransferase by free cholesterol

The administration of mevalonic acid to rats by intravenous injection resulted in a dose- and time-dependent increase in the activity of cholesterol 7α-hydroxylase in the liver microsomal fraction, a decrease in the microsomal activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and no significant change in the activity of acyl-coenzyme A:cholesterol acyltransferase or in the concentration of free and of esterified cholesterol in the liver microsomal fraction. However, the increased hepatic cholesterogenesis that follows the injection of mevalonic acid resulted in an increase of the size of the intracellular pool of cholesterol that is in the environment of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and acts as substrate for cholesterol 7α-hydroxylase. The administration of mevalonic acid to rats by stomach tube resulted in an increase in the activity of cholesterol 7α-hydroxylase and of acyl-coenzyme A:cholesterol acyltransferase and in the concentration of cholesterol esters in the liver microsomal fraction, while there was a considerable decrease in the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Rapid increase in hepatic HMG CoA reductase activity and in vivo cholesterol synthesis after Triton WR 1339 injection.

Triton WR 1339, injected intravenously into rats, caused a 12% decrease in hepatic cholesterol within 30 minutes and a 34% decrease after 2 hours. An early and progressive increase in plasma cholesterol and triglycerides was also confirmed. Although hepatic HMG Coa reductase activity was unchanged after 30 minutes, it had increased seven-fold after 105 minutes. In vivo cholesterol synthesis measured by determining incorporation of intraperitoneally-injected 3H2O into cholesterol also showed an early increase, suggesting that the enzyme was rate controlling for cholesterogenesis under conditions of rapid stimulation. These findings strongly suggest that Triton WR 1339 stimulates hepatic cholesterogenesis by depleting hepatic cholesterol and trapping it in the blood compartment. The rapidity with which the drug acts supports the hypothesis that hepatocellular flux of cholesterol or its derivatized product could mediate the diurnal and hormonally induced fluctuations of cholesterogenesis.

The hypercholesterolaemic effect of caffeine in rats fed on diets with and without supplementary cholesterol.

1. The effect of caffeine (2.5 g/kg diet) on lipid metabolism was examined in rats fed on a stock (low-cholesterol) diet or on a cholesterol plus cholic acid-supplemented (high-cholesterol) semi-synthetic diet. 2. When caffeine was induced in the stock diet fed to rats for 7 d, there was a moderate but significant increase in the concentration of serum cholesterol compared to the levels observed in the control rats. This change can be accounted for by the increase that was observed in the rate of cholesterogenesis in the liver. 3. After 25 d of caffeine in the stock diet, hepatic cholesterogenesis was still increased but the concentration of serum cholesterol was now the same as in the control rats. During the experimental period there was a progressive increase in the faecal excretion of neutral sterols in the rats receiving caffeine. 4. When caffeine was added to a cholesterol plus cholic acid-supplemented diet, there was a marked increase in the concentration of serum cholesterol but hepatic cholesterogenesis was now reduced. 5. Caffeine in the high-cholesterol diet appeared to delay, but probably did not reduce, the absorption of an oral dose of radio-labelled cholesterol. This conclusion was confirmed using rats which had not previously received either caffeine or cholesterol in the diet. 6. When the effect of caffeine in the high-cholesterol diet was investigated during a 24 h period, an exacerbation of the hypercholesterolaemia was seen only at certain times. 7. After a 4-month period of feeding rats on the caffeine-supplemented high-cholesterol diet, histological examination did not detect any damage to the heart and aorta. 8. The metabolic regulations involved in the effects of caffeine in the two diets are discussed and the relevance of the present results to observations made with human subjects is considered.

Comparative effects of amphetamine and fenfluramine on lipid biosynthesis and absorption in the rat

The effects of amphetamine and fenfluramine on lipid biosynthesis and intestinal absorption of triglycerides in the rat were observed independently of the anorectic activity of these drugs. Amphetamine and fenfluramine significantly reduced de novo lipogenesis and cholesterogenesis from 3H 2O and [ 14C] alanine in isolated hepatocytes. Fenfluramine was four times more potent an inhibitor than amphetamine. At high concentrations (above 2 mM), fenfluramine inhibited 14CO 2production from [ 14C] alanine in isolated hepatocytes while amphetamine stimulated 14CO 2 production at all concentrations tested (0.25 to 8 mM). Lipogenesis in liver and adipose tissue was not reduced in vivo by the acute administration of amphetamine. It, however, reduced lipogenesis in the small intestine. Fenfluramine significantly depressed in vivo lipogenesis in liver, small intestine and adipose tissue at 10 and 20 mg/kg, i.p. Hepatic cholesterogenesis was depressed significantly in vivo by both drugs. Fenfluramine increased serum free fatty acids while amphetamine produced no change. Triglycerides were increased significantly by fenfluramine only. Serum cholesterol and phospholipids were unchanged. Fenfluramine at 40 and 60 mg/kg, p.o., diminished significantly the intestinal absorption of triglycerides and depressed the accumulation of lipid in the liver. Analogous effects were not observed with amphetamine. Amphetamine was a weak inhibitor of rat pancreatic lipase (EC 3.1.1.3) ( K i = 21 mM) and fenfluramine was a stronger inhibitor ( K i = 3.3 mM).

URSODEOXYCHOLIC ACID TREATMENT OF GALLSTONES: Dose-response Study and Possible Mechanism of Action

To determine the optimum dose for the medical treatment of gallstones with ursodeoxycholic acid (U.D.C.A.), 11 non-obese patients with radiolucent gallstones were given 5 mg, 10 mg, and 15 mg U.D.C.A. kg body-weight-1 day-1 for 6 weeks each. Apart from 3 patients who required surgery for gallstone complications, treatment was well tolerated, and both dyspeptic symptoms and frequency of biliary colic were reduced. Neither diarrhœa nor hypertransaminasæmia occurred during therapy. Of 3 patients completing 6 months' treatment with an average of 8 mg U.D.C.A. kg-1 day-1, 1 showed complete and 2 partial dissolution of gallstones. There were significant correlations between the biliary cholesterol-saturation index and (i) daily dose of U.D.C.A. in mg kg-1, (ii) percentage U.D.C.A., and (iii) percentage U.D.C.A. + chenodeoxycholic acid (C.D.C.A.) in biliary bile acids. Mean γ-glutamyl transpeptidase fell significantly during treatment, whereas other liver-function tests and hepatic histology remained normal. Mean serum-cholesterol levels did not change, and although there was a 13-35% fall in fasting serum-triglycerides, this difference was not statistically significant. U.D.C.A., like C.D.C.A., seems to act by inhibiting hepatic cholesterogenesis. In 5 patients treated with 5 mg U.D.C.A. kg-1 day-1 for 1-6 months, mean hepatic hydroxymethyl-glutaryl-coenzyme-A-reductase activity was 28% lower than in untreated gallstone patients. These preliminary results suggest that U.D.C.A. is as effective as C.D.C.A. in 1/2—2/3 the dose and that 10 mg U.D.C.A. kg-1 day-1 should be effective in most gallstone patients.

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.

7-Ketocholesterol. Its effects on hepatic cholesterogenesis and its hepatic metabolism in vivo and in vitro.

7-Ketocholesterol. Its effects on hepatic cholesterogenesis and its hepatic metabolism in vivo and in vitro.

Effects of free fatty acids on activity of hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase and on secretion of triglyceride and cholesterol by liver.

The output of triglyceride and cholesterol, and the activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) were measured following perfusion of the isolated rat liver with a medium containing free fatty acids. The activity of the enzyme and the output of cholesterol and triglyceride are regulated by the quantity and structure of the free fatty acid. The activity of HMG-CoA reductase was linearly proportional to uptake of oleic acid (18:1) by the liver. Output of triglyceride and cholesterol in the presence of oleate was also stimulated although, under these experimental conditions, the maximal secretory rate for cholesterol was observed while reductase activity was still increasing. When equimolar quantities of palmitic (16:0), oleic (18:1), or linoleic acid (18:2) were infused, the observed activity of the enzyme was in the order 18:1 greater than 18:2 = 16:0. Output of triglyceride was in the order 18:1 = 18:2 greater than 16:0. Output of free and esterified cholesterol was in the order 18:1 greater than 18:2 greater than 16:0. The stimulation by free fatty acids of hepatic cholesterogenesis, as estimated by the activity of HMG-CoA reductase, probably is the result of stimulation by free fatty acids of secretion of triglyceride in the very low density lipoprotein, and the obligatory requirement for cholesterol as a component of the lipoprotein.

Influence of streptozotocin diabetes on intestinal 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in the rat.

Studies were undertaken to examine cholesterogenesis in the intestine of streptozotocin-diabetic rats by measuring incorporation of [2(-14)C] acetate into cholesterol and 3-hydroxy-3-methylgultaryl CoA reductase (HMG-CoA reductase, EC 1.1.1.34) activity. In these diabetic rats, the intestinal mucosal weight and food consumption were markedly high. The incorporation of [2(-14)C] acetate into cholesterol was significantly increased in all diabetic intestinal segments. However, the rates of production of fatty acids and carbon dioxide were not affected. Hepatic HMG-CoA reductase activities were markedly reduced during both the diurnal high and low periods in these diabetic rats, and there was no diurnal variation. In contrast, the specific activities of this enzyme in jejunal crypt cells during both the diurnal high and low periods were significantly higher in these diabetic rats without loss of diurnal variation. Total reductase activity per segment of intestine in jejunal and ileal mucose (villi + crypt cells) was increased in these diabetic rats. Control rats had higher total and specific activity of ileal mucosal (velli + crypt cells) reductase than of jejunal mucosal reducatse during the diurnal high period. The jejunal-ileal gradient in reductase activity and the incorporation of [2(-14)C] acetate into cholesterol did not change significantly with streptozotocin-diabetic rats. The results indicate that in streptozotocin-diabetic rats, hepatic cholesterogenesis decreases but intestinal synthesis increases.

Effects of Dietary Fiber and Salt Mixtures on the Cholesterol Metabolism of Rats

The isotopic dilution method, which permits the in vivo measurements of the rates of the processes involved in cholesterol turnover, has been applied to rats fed a commercial stock diet or a basal semipurified diet in which either the nature and proportions of the source of dietary fiber or the salt mixture were changed. The cholesterolemia was about 100 mg/100 g in rats fed agar-agar, cellulose, bran or the stock diet. Pectin addition (5%) lowered significantly the plasma concentration of cholesterol (70 mg/100 g). Changes in the source of dietary fiber or salt mixture have moderate effects on the absorption coefficient of dietary cholesterol (range 58.2%-82%). In comparison to agar-agar, cellulose at 2.3% in the diet significantly lowered this coefficient, but larger amounts of cellulose (6.8% or 12.3%), or pectin (5%) were without effect, while bran addition (10%) tended to slightly decrease cholesterol absorption. Hence, high levels of cellulose in the diet increased the absorption coefficient in comparison to a low cellulose diet. A decrease of this coefficient was also observed when the calcium content of the diet was increased. Cholesterol biosynthesis and fecal excretion were inversely correlated to the absorption coefficient of dietary cholesterol in rats fed all of the semipurified diets indicating, as previously shown, that the intestine was the major source of biosynthesized cholesterol diverted into the plasma. However, feeding a commercial stock diet greatly increased the cholesterogenesis and the fecal elimination of bile acids, suggesting a high hepatic cholesterogenesis.

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 rates, low density serum lipoproteins.