Effects of perturbations in hepatic free and esterified cholesterol pools on bile acid synthesis,cholesterol 7α-hydroxylase,HMG-CoA reductase,acyl-CoA:Cholesterol acyltransferase and cytosolic cholesteryl ester hydrolase

Effects of expansion of the hepatic free cholesterol pool on bile acid and cholesterol metabolism and homeostasis were examined in rats fed cholesterol in high-fat diets or treated with oleyl-p-(n-decyl)-benzenesulfonate (ODS) or progesterone. Cholesterol feeding for 10–16 days, which increased free (33%) and esterified (6-fold) cholesterol, had no effect on cholate synthesis, total bile acid synthesis, or cholate turnover, whereas these activities were increased 60–80% by ODS and progesterone, which produced only small increases (19%) in free cholesterol. Cholesterol feeding reduced β-hydroxy-β-methylglutaryl (HMG)-CoA reductase (72%) and cholesteryl ester hydrolase (48%) and increased acyl-CoA:cholesterol acyltransferase (184%), whereas ODS and progesterone reversed these compensatory responses in cholesterol-fed rats. Cholesterol 7α-hydroxylase was changed no more than 22% by any treatment. A bolus of ODS elevated biliary cholesterol output 41% and shifted biliary bile acid synthesis and composition toward 12-deoxy bile acids. These effects were not seen in ODS-fed or progesterone-treated rats, in which cholesteryl ester stores were depleted. It is concluded that effects of free cholesterol on bile acid synthesis and biliary cholesterol are probably mediated by specific precursor or regulatory pools which can be independently regulated and which represent a relatively small fraction of hepatic free cholesterol.     

The effects of simvastatin and cholestyramine,alone and in combination,on hepatic cholesterol metabolism in the male rat

The influence of dietary simvastatin, cholestyramine, and the combination of simvastatin plus cholestyramine on hepatic cholesterol metabolism has been investigated in male rats. Recovery from the effects of the drugs was also investigated by refeeding normal chow for 24 h. Both drugs, alone and in combination, increased 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activityin vitro, but activity returned toward control values, after drug withdrawal. Acyl-CoA:cholesterol acyltransferase (ACAT) was significantly reduced (P<0.001) by simvastatin (−75%), cholestyramine (−71%), and by the drug combination (−81%), due both to a decrease in microsomal cholesterol and to nonsubstrate-dependent modulation of enzyme activity. Refeeding control diet increased ACAT activity but not to control levels. The enhanced activity arose partly from higher microsomal cholesterol and partly from increases in total enzyme activity. Cytosolic neutral cholesteryl ester hydrolase (CEH) activity was substantially elevated by simvastatin (3-fold) and by the drug combination (6-fold), whereas the effect of cholestyramine was smaller (1.5-fold). Normal chow for 24 h only partially returned cytosolic CEH activity to control values. Microsomal CEH activity was increased by simvastatin, alone and in combination with cholestyramine (1.4 to 1.7-fold), and was also enhanced, in the cholestyramine-treated animals, following drug withdrawal. Removal of simvastatin did not allow recovery of this enzyme activity, while withdrawal of the drug combination led to values 29% below controls. The results indicate that in the rat, simvastatin and cholestyramine alter both ACAT and CEH activity, as well as inhibiting HMG-CoA reductase activity.     

Effect of cholesterol and cholestyramine feeding and of fasting on sterol synthesis in the liver,ileum, and lung of the guinea pig

The effects of feeding diets containing either cholesterol (0.24% w/w) or cholestyramine (2.5% w/w) and of fasting on sterol synthesis in the liver, ileum, and lung of both male and female guinea pigs have been studied by measuring the incorporation by tissue slices of¹⁴C-labeled acetate into total digitonin-precipitable sterols. Cholesterol feeding significantly decreased (P<0.05) sterol synthesis in the liver, ileum, and lung of the males and in the ileum of females. Cholestyramine feeding stimulated the rate of hepatic sterol synthesis 13-fold but did not significantly affect sterologenesis in the ileum. Sterol synthesis in the lung was significantly increased (P<0.05) but to a much lesser extent than in the liver. Fatty acid synthesis in the liver, ileum, and lung was not significantly affected by either cholesterol or cholestyramine feeding. In guinea pigs fasted for 24 hr, sterol synthesis was inhibited in all three tissues, the most pronounced effect occurring in the liver. Only in the lung was fatty acid synthesis significantly decreased (P<0.001) by fasting. Cholesterol feeding resulted in increased concentrations of cholesterol in the plasma and liver. Cholestyramine feeding reduced plasma cholesterol concentration by 81% in females and by 64% in males. However, it did not significantly change the tissue cholesterol concentrations. Fasting resulted in a significant increase (P<0.05) in plasma cholesterol concentration but did not affect the concentration of cholesterol in the tissues. It was concluded that in the normal guinea pig, the feedback inhibition produced by both cholesterol and also possibly by bile acids suppresses sterol synthesis in the liver to very low rates compared to those in the small intestine, where sterologenesis is not only less sensitive to the cholesterol negative feedback system than that in the liver, but also is not subject to regulation by the bile acid negative feedback system.     

Effects of cholestyramine on hepatic cholesterol 7α-hydroxylase and serum 7α-hydroxycholesterol in the hamster

Cholestyramine increases activities of hepatic cholesterol 7α-hydroxylase and serum levels of 7α-hydroxycholesterol. To examine if serum 7α-hydroxycholesterol levels parallel with enzyme activity, 0, 0.5, 1, 2, 4, and 10% of cholestyramine was administered to female golden Syrian hamsters for 28 d in the dose-dependent study, and 2% cholestyramine for 0, 1, 3, 7, 14, 21, and 28 d in the time-dependent study. In the dose-dependent study, hepatic and serum cholesterol levels were significantly decreased dose-dependently when more than 0.5% of cholestyramine was fed for 28 d. Cholestyramine increased the cholesterol 7α-hydroxylase activity in a dose-dependent manner, while the serum 7α-hydroxycholesterol level was essentially unchanged. No correlation was found between the serum level and the hepatic enzyme activity. In the time-dependent study, hepatic and serum cholesterol levels markedly decreased when 2% cholestyramine was fed for longer than 3 d. The serum triglyceride level increased significantly for the first 7 d and then decreased. Cholesterol 7α-hydroxylase activity increased significantly as early as day 1, reached maximum activity level on day 7, and then kept the significantly high values until day 28. The serum 7α-hydroxycholesterol level significantly increased for the first 7 d and decreased to the pretreatment level thereafter. 7α-Hydroxycholesterol levels significantly correlated with serum cholesterol and triglyceride levels. We conclude that the serum 7α-hydroxycholesterol level does not always reflect the activity of hepatic cholesterol 7α-hydroxylase, when cholesterol metabolism is severely disturbed by cholestyramine.     

Different thermal behavior of neonatal hepatic and cerebral 3-hydroxy-3-methylglutaryl-CoA reductase

The Arrhenius plots of hepatic and cerebral 3-hydroxy-3-methylglutaryl-CoA reductase activity were studied in neonatal chicks fed with a standard diet. Supplementation of the diet with 2% cholesterol from hatching has no effect on the thermal characteristics of the brain enzyme. The Arrhenius plot of brain reductase was practically similar to that found in control chicks. However, hepatic reductase was inhibited by cholesterol feeding. Dietary cholesterol increased the cholesterol/lipidic phosphorus molar ratio in liver microsomes, whereas no significant differences were observed in brain microsomes. These results are in agreement with the hypothesis that activity of hepatic reductase is regulated by the fluidity of microsomal membrane and show that cholesterol feeding does not alter the fluidity of microsomal membranes in neonatal chick brain having, thus, no effect on the thermal behavior of cerebral reductase.     

Effect of candicidin on cholesterol and bile acid metabolism in the rat

Sterol metabolism studies were carried out in rats maintained on a diet containing a polyene antibiotic, candicidin, (30 mg/kg/day) for 2-1/2 months. Compared to the controls, the candicidintreated animals had a smaller food intake and weight gain during this period. There was no difference between the 2 groups in serum cholesterol levels, biliary cholesterol or bile acid concentrations. However, in the experimental group, liver cholesterol content decreased by 27% and hepatic HMG-CoA reductase increased by 36%. Candicidin administration produced an 84% increase in neutral sterol output without change in bile acid output. Cholesterol absorption was reduced 80% by candicidin feeding. The weight of ventral prostate was reduced 33% by candicidin administration. Prostatic HMG-CoA reductase levels were 3 times higher than those of the liver, but enzyme activity was unchanged by candicidin treatment.     

Secondary regulatory sites in rat liver cholesterol biosynthesis: Role of 5-pyrophosphomevalonate decarboxylase

The activity of 5-pyrophosphomevalonate decarboxylase in 43,000 g supernatant fractions from livers and kidneys of male adult rats has been determined. Enzyme activity in liver is significantly increased when rats are fed a diet containing 3% cholestyramine (268% of control rats) and decreased when fed a diet containing 2% cholesterol (25% of control rats). No circadian rhythm of enzyme activity is found in liver or kidneys. These results show that variations in hepatic cholesterogenesis affect the activity of 5-pyrophosphomevalonate decarboxylase in a similar way as other enzymes involved in the biosynthesis of cholesterol.     

Resistant starch is more effective than cholestyramine as a lipid-lowering agent in the rat

Amylase-resistant starch (RS) represents a substrate for the bacterial flora of the colon, and the question arises as whether RS shares with soluble fibers common mechanisms for their lipid-lowering effects. It is uncertain whether a cholesterol-lowering effect depends basically on an enhanced rate of steroid excretion or whether colonic fermentations also play a role in this effect. In the present study, the effect of RS (25% raw potato starch), of a steroid sequestrant (0.8% cholestyramine), or both were compared on bile acid excretion and lipid metabolism in rats fed semipurified diets. RS diets led to a marked rise in cecal size and the cecal pool of short-chain fatty acids (SCFA), as well as SCFA absorption; cholestyramine did not noticeably affect cecal fermentation. Whereas cholestyramine was particularly effective at enhancing bile acid excretion, RS was more effective in lowering plasma cholesterol (−32%) and triglycerides (−29%). The activity of 3-hydroxy-3-methylglutaryl-CoA reductase was increased fivefold by cholestyramine and twofold by RS. This induction in rats fed RS diets was concomittant to a depressed fatty acid synthase activity. In rats fed the RS diet, there was a lower concentration of cholesterol in all lipoprotein fractions, especially the (d=1.040−1.080) fraction high-density lipoprotein (HDL₁), while those fed cholestyramine had only a significant reduction of HDL₁ cholesterol. In contrast to cholestyramine, RS also depressed the concentration of triglycerides in the triglyceride-rich lipoprotein fraction. There was no noticeable synergy between the effects of RS and cholestyramine when both were present in the diet. This suggests that the cholesterol-lowering effect of RS is not limited to its capacity to enhance bile acids excretion. The difference between RS and cholestyramine could relate to the capacity of fermentation end-products to counteract the upregulation of cholesterol and bile acid biosynthesis. Thus, in the absence of fermentation in the large intestine, a high rate of bile acids excretion is not always sufficient to elicit a cholesterol-lowering effect.     

The cholesterol-lowering effect of guar gum in rats is not accompanied by an interruption of bile acid cycling

A viscous hydrocolloid (guar gum, GG; 2.5% of the diet) or a steroid sequestrant (cholestyramine; 0.5% of the diet) was included in semipurified diets containing 0.2% cholesterol to compare the cholesterol-lowering effects of each agent in rats. In the present model, GG significantly lowered plasma cholesterol (−25%), especially in the density <1.040 kg/L fraction, whereas cholestyramine was less potent. Bile acid fecal excretion significantly increased only in rats fed cholestyramine, similar to the cecal bile acid pool; the biliary bile acid secretion was accelerated by GG, but not their fecal excretion, whereas GG effectively enhanced neutral sterol excretion. As a result, the total steroid balance (+13 μmol/d in the control) was shifted toward negative values in rats fed the GG or cholestyramine diets (−27 or −50 μmol/d, respectively). Both agents induced liver 3-hydroxy-3-methylglutaryl-CoA reductase, but cholestyramine was more potent than GG in this respect. The present data suggest that, at a relative low dose in the diet, GG may be more effective than cholestyramine in lowering plasma cholesterol by impairing cholesterol absorption and by accelerating the small intestine/liver cycling of bile acids, which is interestingly, accompanied by reduction of bile acid concentration in the large intestine.     

Effect of lipid content of diet on cholesterol content and cholesterogenic enzymes of European eel liver

The effect of dietary lipid levels on the levels of cholesterol and the activities of the major cholesterogenic enzymes of the liver has been studied in the European eel. An increase in hepatic total cholesterol was observed when the dietary lipid levels increased from 12 to 20%, while protein levels were maintained at 30%. This change paralleled an increase in mevalonate 5-pyrophosphate decarboxylase activity, while 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase and mevalonate 5-phosphate kinase were not affected by changes in diet composition. These results suggest that the decarboxylase may be a rate-limiting enzyme in cholesterogenesis in eel liver.     

Age-related changes in the lipid metabolism of Fisher 344 rats

Lipid metabolism of male Fisher 344 rats aged 2–24 months was studied. Serum and liver cholesterol levels did not display the age-related gradual increase seen in other rat strains. An increase in the serum plus liver cholesterol pool from 2 to 6 months was followed by a plateau through 18 months and then another increase at 24 months of age. The triglyceride pool increased from 2 to 6 months and then remained unchanged through 24 months of age. Cholesterol synthesis from acetate decreased 50% between 2 and 9 months and fell only slightly through 24 months of age. Assay of 3-hydroxy-3-methyl glutaryl Coenzyme A (HMG-CoA) reductase showed a similar pattern but did not decrease further after 9 months of age. Cholesterol 7α hydroxylase activity was not significantly altered by age. These age- and strain-related differences present an opportunity for a comparative study of the aging process using the parameters of lipid metabolism as indicators.     

Acyl-CoA:cholesterol acyltransferase activity in the rat mammary gland: Variation during pregnancy and lactation

Cholesterol esterification by acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) has been studied in microsomes isolated from the mammary glands of rats in late pregnancy, in early and mid-lactation, and following premature weaning. The mammary glands were freeze-clamped and the microsomes prepared in the presence of phosphatase inhibitors to preserve the phosphorylation status of the enzyme. Optimal conditions were established for the assay of enzyme activity in the presence of endogenous cholesterol. Supplementation of the microsomes with exogenous cholesterol as a dispersion in Triton WR-1339 was shown to lead to an increase in enzyme activity. Incubation of microsomes with MgATP led to an increase in ACAT activity which could be reversed by treatment of the microsomes with a phosphoprotein phosphatase preparation from rat liver. The results suggested that ACAT activity in the mammary gland was activated by phosphorylation in a similar way to that observed for the hepatic enzyme. The mammary glands from pregnant animals contained a higher level of ACAT activity than did the glands of the lactating animals and this correlated with the higher cholesteryl ester content of the pregnant glands. The highest level of ACAT activity was found in the weaned animals but the cholesteryl ester content of the microsomes was lower than expected. The influence of progesterone levels and changes in feeding patterns during gestation were considered as factors in these variations in ACAT activity.     

Eicosapentaenoic acid,but not docosahexaenoic acid,increases mitochondrial fatty acid oxidation and upregulates 2,4-dienoyl-CoA reductase gene expression in rats

The aim of the present study was to investigate whether eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) was responsible for the triglyceride-lowering effect of fish oil. In rats fed a single dose of EPA as ethyl ester (EPA-EE), the plasma concentration of triglycerides was decreased at 8 h after acute administration. This was accompanied by an increased hepatic fatty acid oxidation and mitochondrial 2,4-dienoyl-CoA reductase activity. The steady-state level of 2,4-dienoyl-CoA reductase mRNA increased in parallel with the enzyme activity. An increased hepatic long-chain acyl-CoA content, but a reduced amount of hepatic malonyl-CoA, was obtained at 8 h after acute EPA-EE treatment. On EPA-EE supplementation, both EPA (20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3) increased in the liver, whereas the hepatic DHA (22:6n-3) concentration was unchanged. On DHA-EE supplementation retroconversion to EPA occurred. No statistically significant differences were found, however, for mitochondrial enzyme activities, malonyl-CoA, long-chain acyl-CoA, plasma lipid levels, and the amount of cellular fatty acids between DHA-EE treated rats and their controls at any time point studied. In cultured rat hepatocytes, the oxidation of [1-¹⁴C]palmitic acid was reduced by DHA, whereas it was stimulated by EPA. In thein vivo studies, the activities of phosphatidate phosphohydrolase and acetyl-CoA carboxylase were unaffected after acute EPA-EE and DHA-EE administration, but the fatty acyl-CoA oxidase, the rate-limiting enzyme in peroxisomal fatty acid oxidation, was increased after feeding these n-3 fatty acids. The hypocholesterolemic properties of EPA-EE may be due to decreased 3-hydroxy-3-methylglutaryl-CoA reductase activity. Furthermore, replacement of the ordinary fatty acids, i.e., the monoenes (16:1n-7, 18:1n-7, and 18:1n-9) with EPA and some conversion to DPA concomitant with increased fatty acid oxidation is probably the mechanism leading to changed fatty acid composition. In contrast, DHA does not stimulate fatty acid oxidation and, consequently, no such displacement mechanism operates. In conclusion, we have obtained evidence that EPA, and not DHA, is the fatty acid primarily responsible for the triglyceride-lowering effect of fish oil in rats.     

The hypolipidemic activity of boronated nucleosides in male mice and rats

The boronated nucleosides with varying bases and sugar moieties were shown to be potent hypolipidemic agents in rodents. The 3'- aminocynaoborane dideoxythymidine derivative caused reductions in serum cholesterol and triglyceride levels, tissue lipids, VLDL and LDL cholesterol levels while elevating HDL cholesterol levels in rodents. The agents suppressed rat hepatic acetyl CoA synthetase, HMG-CoA reductase, acyl-CoA cholesterol acyl transferase, phosphatidylate phosphohydrolase and lipoprotein lipase activities while elevating cholesterol-7alpha-hydroxylase activity from 25 to 100 muM.     

Mevalonate 5-pyrophosphate decarboxylase in isolated villus and crypt cells of chick intestine

Mevalonate 5-pyrophosphate decarboxylase was studied in isolated enterocytes obtained from duodenal, jejunal and ileal villi and crypts. In our assay conditions, decarboxylase activity was linear for 60 min and up to 0.3 mg of protein. The subcellular location of decarboxylase in chick enterocytes was investigated. About 94% of the total activity was recovered in the cytosol. The distribution of enzyme activity in epithelial cells also was studied. Maximal specific activity was found in cell fractions from jejunum followed by ileum and duodenum. About 80% of total activity was recovered in the villus cells, indicating an active role of these cells in cholesterogenesis. Ileal cells showed the highest cholesterol content. In all the intestinal epithelial cells assayed, free cholesterol represented about 95% of the total cholesterol.     

Cholesterol metabolism in frog (Rana esculenta) liver: Seasonal and sex-related variations

Many aspects of lipid metabolism have been studied in amphibians, but seasonal lipid modulation in male and female frogs has not been investigated. We describe here the yearlong patterns of hepatic lipid content and enzyme activities related to cholesterol homeostasis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and acyl coenzyme A:cholesterol acyltransferase (ACAT) activity in liver of the male and female frog,Rana esculenta. Lipid storage follows distinct seasonal patterns, with an increase in June that is more pronounced in the female than in the male frog. Cholesterol content and cholesterol storage as cholesteryl ester in male liver are consistent with the activity of HMG-CoA reductase and of ACAT enzymes. HMG-CoA reductase activity of the female frog shows an extra peak in fall unrelated to cholesterol storage and probably related to the production of essential compound for oogenesis.     

The effects of boron derivatives on lipid absorption from the intestine and on bile lipids and bile acids of sprague dawley rats

N,N-dimethyl-n-octadecylamine borane 1 at 8 mg/kg/day, tetrakis-u-(trimethylamine boranecarboxylato)-bis(trimethyl-carboxyborane)-dicopper(II) 2 at 2.5 mg/kg/day and trimethylamine-carboxyborane 3 at 8 mg/kg/day were evaluated for their effects on bile lipids, bile acids, small intestinal absorption of cholesterol and cholic acid and liver and small intestinal enzyme activities involved in lipid metabolism. The agent administered orally elevated rat bile excretion of lipids, e.g. cholesterol and phospholipids, and compounds 2 and 3 increased the bile flow rate. These agents altered the composition of the bile acids, but there was no significant increase in lithocholic acid which is most lithogenic agent in rats. The three agents did decrease cholesterol absorption from isolated in situ intestinal duodenum loops in the presence of drug. Hepatic and small intestinal mucosa enzyme activities, e.g. ATP-dependent citrate lyase, acyl CoA cholesterol acyl transferase, cholsterol-7-alpha -hydroxylase, sn glycerol-3-phosphate acyl transferase, phosphatidylate phosphohydrolase, and lipoprotein lipase, were reduced. However, the boron derivatives 1 and 3 decreased hepatic HMG-CoA reductase activity, the regulatory enzyme for cholesterol synthesis, but the compounds had no effects on small intestinal mucosa HMG-CoA reductase activity. There was no evidence of hepatic cell damage afforded by the drugs based on clinical chemistry values which would induce alterations in bile acid concentrations after treatment of the rat.     

Effects of cholestanol feeding and cholestyramine treatment on the tissue sterols in the rabbit

Rabbits were fed diets enriched with cholestanol or cholestereol (3.5 g/wk) for 4–12 weeks. During cholestanol feeding, the concentration of cholestanol in blood serum, liver, heart and aorta increased 15–30 times. In serum and liver, the concentration of cholesterol also increased. Cholestanol-fed rabbits developed inflammatory changes in the liver, with proliferation of small bile ducts. Liver tests were only slightly abnormal. Morphological atherosclerosis of the aorta was only occasionally seen in rabbits receiving cholestanol for eight weeks or less. During cholesterol feeding, the amounts of cholesterol in different tissues increased dramatically, most in the aorta. Morphological atherosclerosis in the aorta was found in all rabbits fed cholesterol-enriched diets for more than four weeks. Brain cholestanol was doubled in rabbits fed cholestanol for eight weeks, whereas brain sterols did not change significantly during cholesterol feeding. After an additional regression period with cholestyramine for eight weeks, the increased content of cholestanol in the brain was unchanged in cholestanol-fed rabbits. These observations are discussed in relation to the cholestanolosis of the brain that develops in the rare inherited human disease cerebrotendinous xanthomatosis.     

Effect of type of diet and feeding status on modulation of hepatic HMG-CoA reductase in rats

The effect of diet type and feeding status on hepatic HMG-CoA reductase (HMGR) [mevalonate: NADP⁺ oxidoreductase (acylating CoA); EC 1.1.1.34] was studied in rats. Animals fed a ground, commercial, stock diet exhibited higher expressed and total activities of HMGR in the fed state than animals fed a semi-purified diet. The differences did not appear in meal-trained animals when measured before the onset of the meal after a 22-hr fast. When expressed activity was taken as a per cent of total activity, fed animals from both diet groups used about 10% of their available activity. When animals on commercial diets were fasted, 20% of the activity was expressed. Fasted animals on the semi-purified diet also increased the per cent of expressed reductase activity, but this increase was not as great (13.3%). These data suggest that, in the rat, regulation of cholesterol synthesis in response to decreased total HMGR during fasting and increased levels after a meal results from alterations in the percentage of enzyme which is expressed. The semi-purified diet used here resulted in consistently lower levels of HMG-CoA reductase activity than the commercial diet regardless of feeding pattern.