Effects of colestipol hydrochloride and neomycin sulfate on cholesterol turnover in the rat

Three groups of male rats were fed diets containing the bile acid sequestrant colestipol hydrochloride (1%), neomycin sulfate (0.25%), or basic diet during the test. After 15 days, each rat was injected IV with 3.9 μCi cholesterol-1,2-³H complexed with serum lipoproteins; specific radioactivity of the total serum cholesterol was measured at several time intervals for a period of 7 weeks. Computer analysis of the data indicated that the turnover of cholesterol could best be fitted by a three-pool model. In pool 1, colestipol HCl caused a significant increase in production rate (10.09 to 15.96 mg/day) and the excretion rate constant (0.53 to 0.79 day⁻¹) of cholesterol without significantly altering the size of the pool or serum cholesterol concentrations. These results are compatible with an agent capable of binding bile acids in the rat but do not cause a decrease of the sterol pool because of an adequate compensatory increase in cholesterol biosynthesis. Neomycin SO₄ caused a significant reduction in serum cholesterol (9%) without altering turnover parameters and apparently exerts its hypocholesterolemia by some mechanism other than bile acid sequestration.     

Effects of ketoconazole on cholesterol synthesis and precursor concentrations in the rat liver

Ketoconazole, an antimycotic agent, given to rats for a week as 0.05% food addition had no effect on the hepatic concentrations of free and esterified cholesterol or on the activity of acyl coenzyme A: cholesterol-acyltransferase (ACAT). However, the levels of free methylated cholesterol precursors, especially lanosterols, less markedly Δ^8,24 and Δ⁸-dimethyl sterols and monomethyl sterols, were increased after only one day's treatment, while those of esterified methyl sterols were increased inconsistently, and those of free and esterified Δ⁸-lathosterol, lathosterol and desmosterol were not affected at all. Cholestyramine treatment had no significant effect on ACAT in spite of a decrease in the hepatic content of esterified cholesterol and caused a marked increase in the free cholesterol precursor levels, especially in those of lathosterols. Cholestyramine given to ketoconazole-treated rats increased the hepatic levels of Δ⁸ and Δ⁷-lathosterols but not desmosterol or methylated cholesterol precursors. Ketoconazole increased and cholestyramine markedly decreased plantssterols, sitosterol and campesterol in the liver. In serum, the contents of both lanosterols and lathosterol were increased but that of cholesterol tended to be decreased by ketoconazole (−19%). The results indicate that ketoconazole impairs demethylation processes at C-14 and to some extent at C-4 in the rat liver, resulting in lowered serum cholesterol level.     

Effects of sodium glycocholate and sodium taurocholate on the mixed micelles of bile salts and nonionic surfactant

Effects of sodium glycocholate (NaGC) and sodium taurocholate (NaTC) on the mixed micelles for two systems consisting of NaGC-octaoxyethylene glycol monon-decyl ether (C₁₀E₈) and NaTC-C₁₀E₈ have been studied as a function of the mixed micelles’ compositions, polarities of the micelles’ interior and mean aggregation numbers. The compositions of the mixed micelles are calculated from critical micelle concentration (CMC) data by using excess thermodynamic quantities. The polarities and mean aggregation numbers are determined from pyrene fluorescence in the mixed micelles. Both mixed systems were nonideal, and the mole fraction of NaGC or NaTC in a mixed micelle near the CMC was less than that in the aqueous mixed solution. However, the mixed micelle of the NaTC-C₁₀E₈ system contained more bile salt molecules than that of the NaGC-C₁₀E₈ system because of a good miscibility of NaTC and C₁₀E₈ molecules. The pyrene fluorescence results suggested that the mixed micelles changed from C₁₀E₈-rich micelles to NaGC- or NaTC-rich micelles, and mean aggregation numbers of the mixed micelles decreased abruptly with increasing mole fraction of bile salts. In the low mole fraction range of bile salts, however, both the polarities and the mean aggregation numbers for the NaTC-C₁₀E₈ system are lower than those for the NaGC-C₁₀E₈ system because of the high mole fraction of NaTC in a mixed micelle, and also because of the different effect of the conjugated group between NaTC and NaGC molecules in the mixed micelles.     

The effect of a non-absorbable fat on the turnover of plasma cholesterol in the rat

Rats were injected with [4-¹⁴C]-cholesterol and then fed diets that contained sucrose polyester (SPE) at levels of 0 and 8% of the diet.¹⁴C was measured in neutral and acidic steroid fractions of the feces collected during days 35–39 post i.v. injection. Periodic blood samples were used to measure the specific activity of the plasma cholesterol. The plasma data were consistent with a two-pool model for the decay of the plasma specific activity. The slow component of the decay curve decreased more rapidly in animals that received SPE. The half-life corresponding to this component was approximately 20% shorter in the SPE-fed animals compared to the control group. The mass of cholesterol calculated for the first pool was similar for all groups of animals. The¹⁴C found in the feces was consistent with the more rapid removal of cholesterol from the body in the SPE-fed animals. The mass of excreted steroid was equal to the calculated rate of cholesterol production in each group of animals.     

Effect of n−3 fatty acids on the key enzymes involved in cholesterol and triglyceride turnover in rat liver

The effect of long-chain n−3 fatty acids on hepatic key enzymes of cholesterol metabolism and triglyceride biosynthesis was investigated in two rat models. In the first model, rats were intravenously infused for two weeks with a fat emulsion containing 20% of triglycerides in which either n−6 or n−3 fatty acids predominated. The treatment with n−3 fatty acids led to a reduction primarily of serum cholesterol (45%), but also of serum triglycerides (18%). HMG-CoA reductase activity and cholesterol 7α-hydroxylase activity were reduced by 45% and 36%, respectively. There were no significant effects on diacylglycerol acyltransferase (DGAT) or phosphatidate phosphohydrolase (PAP) activities. In the second model, rats were fed a diet enriched with sucrose, coconut oil and either sunflower oil (n−6 fatty acids) or fish oil (long-chain n−3 fatty acid ethyl esters). The treatment with n−3 fatty acids decreased serum triglycerides (41%) and, to a lesser extent, serum cholesterol (17%). Neither glycerol 3-phosphate acyltransferase (GPAT) or DGAT were affected by n−3 fatty acids. In contrast, PAP activity was reduced by 26%. HMG-CoA reductase was not significantly affected, whereas cholesterol 7α-hydroxylase activity was reduced by 36%. The results indicate that part of the TG-lowering effect of long-chain n−3 fatty acids may be mediated by inhibition of the soluble phosphatidate phosphohydrolase. The effect on serum cholesterol may be partly due to inhibition of HMG-CoA reductase.     

The turnover time of dietary cholesterol in the lactating rat

The secretion of dietary 4-¹⁴C-cholesterol into milk of the rat was determined as a function of post-feeding time by a single dose technique. The time interval which elapsed before maximum specific radioactivity was reached in milk (17–20 hr after maximum activity in the serum) suggests a route through the mammary gland involving transport of the cholesterol by intracellular membranes. It also suggests that the exogenous cholesterol is incorporated into the milk fat globule membranes rather than into the fat globules during their synthesis within the cell. Paper No. 3978 in the Journal Series of the Pennsylvania Agricultural Experiment Station.     

Effects of pregnenolone-16α-carbonitrile on the metabolism of cholesterol in rat liver microsomes

The effects of pregnenolone-16α-carbonitrile (PCN) on hepatic metabolism of cholesterol were studied in rat liver microsomes in order to clarify the underlying mechanisms of the PCN-induced biliary hypersecretion of cholesterol. Male Sprague-Dawley rats were fed a diet supplemented with 0.05% of PCN for one week. The microsomal activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, regulating cholesterol biosynthesis, decreased from 577 ± 46 (SEM) to 367 ± 38 pmol/min/mg protein compared to the controls. Cholesterol 7 α-hydroxylase activity, governing bile acid synthesis, was 9.0 ± 1.1 pmol/min/mg protein in the treated group and 34.8 ± 7.4 pmol/min/mg protein in the controls, a reduction of 74% (P<0.01). The acyl CoA:cholesterol acyltransferase (ACAT) activity, catalyzing the esterification of cholesterol, remained unchanged, as did the levels of total and free cholesterol in liver homogenates and microsomes. The results of this study provide evidence that the increase in biliary cholesterol secretion during PCN treatment is not caused by a change in ACAT activity, but can be explained by a decreased catabolism of cholesterol to bile acids.     

Plasma kinetics of free and esterified cholesterol in familial hypercholesterolemia: Effects of simvastatin

The objective of this study was to evaluate the kinetics of both free and esterified forms of cholesterol contained in a emulsion that binds to LDL receptors (LDE) in subjects with heterozygous familial hypercholesterolemia (FH), and the same subjects under the effects of high-dose simvastatin treatment, as compared with a control normolipidemic group (NL). Twentyone FH patients (44.0±13.0 yr, 12 females, LDL cholesterol levels 6.93±1.60 mmol/L) and 22 normolipidemic patients (44.0±15.0, 10 females, LDL cholesterol levels 3.15±0.62 mmol/L) were injected intravenously with ¹⁴C-cholesteryl ester and ³H-cholesterol. FH patients were also evaluated after 2 mon of 40 or 80 mg/d simvastatin treatment, and plasma samples were collected over 24 h to determine the residence time (RT, in h) of both LDE labels, expressed as the median (25%; 75%). The RT of both ¹⁴C-cholesteryl ester and ³H-cholesterol were greater in FH than in NL [FH: 36.0 (20.5; 1191.0), NL: 17.0 (12.0–62.5), P=0.015; and FH: 52.0 (30.0; 1515.0); NL 20.5 (14.0–30.0) P<0.0001]. Treatment reduced LDL cholesterol by 36% (P<0.0001), RT of ¹⁴C-cholesteryl ester by 49% (P=0.0029 vs. baseline), and ³H-cholesterol RT by 44% (P=0.019 vs. baseline). After treatment, the RT values of ¹⁴C-cholesteryl ester in the FH group approached the NL values (P=0.58), but the RT of ³H-cholesterol was still greater than those for the NL group (P=0.01). The removal of LDE cholesteryl esters and free cholesterol was delayed in FH patients. Treatment with a high dose of simvastatin normalized the removal of cholesterol esters but not the removal of free cholesterol.     

Effect of ingestion of isomeric fatty acids on cholesterol and lipids of serum and liver

The effect of the ingestion of large amounts of thetrans, trans isomer of linolein upon the cholesterol and lipid levels of the blood and liver was investigated using hypercholesterolemic rats. The serum levels of esterified fatty acids and cholesterol of rats fed the diets containing 30% oftrans, trans linoleic acid glycerides and safflower oil were 15 and 25%, respectively, lower than those fed coconut oil. However, a weight loss associated with less intake of thetrans isomer as compared with a gain with the other two fats must be considered. The serum levels of labeled cholesterol of rats administered radioactive cholesterol along with thetrans isomer were intermediate in maximum value as compared to the levels obtained with coconut and safflower oils. These results suggest that thetrans isomers are not as effective as thecis isomers in lowering the cholesterol and lipids of the blood. The livers of the former group had a lower fat content than the latter which might be accounted for in part by the lower fat intake Presented at AOCS Meeting, Houston, Texas, 1965.     

Plasma cholesterol turnover and esterification in the pigeon

The plasma cholesterol turnover and serum cholesterol esterifying system was studied in White Carneau pigeons. Eight pigeons received a single injection of 1,2-³H-cholesterol intravenously and the decline in plasma cholesterol specific activity was measured at intervals from 1–64 days. Kinetic analysis of the plasma cholesterol die-away curves indicates that plasma cholesterol turnover in the pigeon conforms to a 2 pool model. The mass of pool A (cholesterol in blood and those tissues which are rapidly equilibrated with blood) in pigeons maintaining consistently high serum cholesterol levels (≈900 mg/100 ml) was 988 mg and the daily fractional turnover of pool A was 19.7% compared with 676 mg and 12.2% found in pigeons maintaining consistently low (≈400 mg/100 ml) serum cholesterol levels. Serum cholesterol esterifying activity, in vitro, showed a positive correlation (rxy=0.806) with serum cholesterol concentration in the pigeon. The pigeon differs, in this regard, from the chicken, rat and rabbit in which a negative correlation has been reported.     

The effects of phosphate on the biosynthesis of cholesterol in rat liver homogenates

The biosyntheses of cholesterol from acetate and mevalonate were determined in rat liver homogenates that were prepared and incubated in buffers containing varying concentrations of phosphate. Relatively little acetate or mevalonate was incorporated into cholesterol in the absence of added phosphate. When phosphate was added, there was an increase in incorporation of both substrates. The addition of phosphate resulted in an increase in the incorporation of mevalonate to a maximum, whereas phosphate appeared to increase the incorporation of acetate at low phosphate levels and decrease the incorporation at higher phosphate levels. The results appear to be consistent with the possibility that, at low phosphate levels, the biosynthesis of cholesterol is limited by some phosphaterequiring reaction(s) in the pathway after mevalonate, and at higher phosphate levels, the biosynthesis is limited by the 3-hydroxy-3-methylglutaryl coenzyme A reductase-catalyzed step.     

Age-related changes in the metabolism of cholesterol in rat liver microsomes

The effects of aging on the hepatic metabolism of cholesterol were studied in 1-, 6- and 24-month-old male Sprague-Dawley rats. Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, which regulates cholesterol biosynthesis, decreased from 835±144 (SEM) pmol/min/mg protein in the youngest group to 219±34 and 205±53 pmol/min/mg protein (p<0.001) in the 6- and 24-month-old groups, respectively. Cholesterol 7α-hydroxylase activity, which governs bile acid synthesis, was gradually reduced from 70±14 pmol/min/mg protein in the 1-month-old group to 32±7 and 16±3 pmol/min/mg protein (p<0.05) in the 6- and 24-month-old groups, respectively. Acyl coenzyme A:cholesterol acyltransferase activity, which catalyzes the esterification of cholesterol, averaged 431±47 and 452 ±48 pmol/min/mg protein in the 1- and 6-month-old groups, respectively, and was increased to 585±55 pmol/min/mg protein (p<0.05) in the 24-month-old group. The level of total cholesterol showed an age-related increase from 1.56±0.16 mg/g liver in the 1-month-old group to 1.70±0.15 and 2.20±0.19 mg/g liver (p<0.05) in the 6- and 24-month-old groups, respectively. The increase was mainly caused by an accumulation of esterified cholesterol. We conclude that a marked decrease in HMG-CoA reductase occurs between 1 and 6 months of age; thereafter the enzyme activity stays unchanged. The activity of cholesterol 7α-hydroxylase decreases progressively and drastically with age, whereas the capacity for esterifying cholesterol increases slightly. We speculate that the reduced conversion of cholesterol to bile acids may be one explanation of the age-related increase of plasma cholesterol seen in rats.     

Uptake and metabolism of dolichol and cholesterol in perfused rat liver

The uptake of dolichol and cholesterol by perfused rat liver was studied. When these radioactive lipids were incorporated into egg phosphatidylcholine liposomes, both dolichol and cholesterol appeared initially in the supernatant and in the microsomal fraction and, later on, in the mitochondrial-lysosomal fraction. The lipids taken up were esterified to some extent, but no phosphorylation of dolichol occurred. Incorporation of dolichol and cholesterol into lipoproteins increased the efficiency of uptake, which was receptor-mediated in this case. Accumulation of these lipids occurred in lysosomes followed by a transport to the endoplasmic reticulum (ER). Both labeled dolichol and cholesterol appeared in the bile. In the case of dolichol, the majority of this radioactivity was not associated with the original substance itself, and probably represented lipid-soluble catabolites. In the case of cholesterol, most of the radioactivity was associated with bile acids. It appears that, in contrast to the receptor-mediated uptake of lipoproteins from the perfusate, the uptake of liposomal lipids involves a different mechanism. After association with the plasma membrane, the lipids enter into the cytoplasm and are transported to the ER and later to the lysosomes.     

The effects of clofibrate and bezafibrate on cholesterol metabolism in the liver of the male rat

Fibric acid derivatives are used to treat hyperlipidemia and have wide ranging effects on lipid metabolism. The action of these compounds on cholesterol esterification, catalyzed by acyl-coenzyme A:cholesterol acyltransferase (ACAT), has been quite widely studied, but their effect on cholesteryl ester hydrolysis and the enzyme neutral cholesteryl ester hydrolase (nCEH) has been largely ignored. Male rats were therefore fed for 10 d on a standard chow diet supplemented with either clofibrate or bezafibrate, to study their effects on plasma lipid levels and hepatic cholesterol metabolism. Plasma triacyglycerols were not significantly altered by these diets, but bezafibrate significantly lowered plasma cholesterol levels (29.7%,P<0.01). When expressed per unit weight of DNA, both fibrates reduced the hepatic content of triacylglycerol, cholesterol and cholesteryl esters (40, 18.7, 16.5 and 66.7, 28.6, 34.2% for clofibrate and bezafibrate, respectively). ACAT activity was significantly reduced by both drugs, but clofibrate (65% inhibition) was more effective than bezafibrate (35% inhibition). The most dramatic effect of the diets was a marked increase in the activity of both the microsomal and the cytosolic nCEH. When expressed on a whole liver basis, the effect of bezafibrate on the cytosolic enzyme (13.6-fold increase in activity) was much greater than that of clofibrate (4.8-fold increase). Increases in the activity of a cytosolic protein that inhibits the activity of nCEH were also noted, but these changes were relatively small. The results suggest that the activation of nCEH, in combination with the inhibition in ACAT activity, contributes to a decrease in the cholesteryl ester content of the liver which may influence the secretion of very low density lipoprotein.     

Effects of oxidative stress on glycerolipid acyl turnover in rat hepatocytes

Lipid peroxidation was induced in freshly isolated rat hepatocytes by incubation in the presence of Fe³⁺, resulting in accumulation of thiobarbituric acid reactive substances. Analysis of lipid classes revealed that the levels and fatty acid compositions of the two major phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), remained unchanged but the levels of triacylglycerols (TAG) were significantly reduced, and some of their polyunsaturated fatty acids were selectively lost as the result of oxidant treatment. Acyl turnover in PC and PE as determined by ¹⁸O incorporation from H₂ ¹⁸O-containing media remained largely unchanged during oxidant treatment, while some increased turnover of the saturated fatty acids in TAG was observed. We hypothesize that constitutive recycling of membrane phospholipids rather than selective in situ repair eliminates peroxidized species of PC and PE, TAG could serve as an expendable fatty acid reserve, providing a limited but very dynamic pool of polyunsaturated fatty acids for the resynthesis of phospholipids.     

Effects of peroxidative stress and age on the concentration of a deoxyguanosine-malondialdehyde adduct in rat DNA

The effect of age and peroxidative stress on the concentration of a deoxyguanosine malondialdehyde adduct (dG-MDA) in rat tissues was investigated. Vitamin E deficiency had not effect on the dG-MDA content of liver DNA in rats fed a diet containing 10% corn oil. When 2% cod liver oil was added to this diet, the dG-MDA content of liver DNA doubled in the positive controls fed a high level of vitamin E (100 ppm dl-α-tocopherol), and there was a further increase when vitamin E was deleted. Neither iron nitrilotriacetate administration nor choline deficiency had any effect on the dG-MDA content of liver DNA. Carbon tetrachloride had a lowering effect. The failure of iron or carbon tetrachloride administration and of vitamin E deficiency to increase liver dG-MDA is consistent with their failure in previous experiments to affect the urinary excretion of dG-MDA. In contrast, these forms of peroxidative stress produce large increments in the urinary excretion of MDA adducts with lysine, reflecting increased formation and degradation of MDA-modified proteins. DNA appears to be protected from modification by MDA produced at extranuclear sites. The frequency of dG-MDA in different tissues of 4-month-old rats varied markedly: brain ≫ liver > kidneys and testes. Higher concentrations of dG-MDA were found in the liver and kidneys, but not the testes, of 25-month-old rats. The determinants of the concentration of dG-MDA in DNA merit further investigation.     

Effects of renal factors on in vitro hepatic cholesterol synthesis in the rat

Two products derived from rat renal tissue have been shown to affect in vitro hepatic cholesterol synthesis. A premevalonate inhibitor of hepatic cholesterol synthesis is associated with the membranes of the renal endoplasmic reticulum. It is stable at −75 C and at 4 C but is heat labile. A pre-mevalonate stimulator of in vitro hepatic cholesterol synthesis is located within renal lysosomes and can be prepared in a nonsedimentable form by extraction with hypotonic buffer. While it is stable at −75 C, it loses activity at 4 C. Both of these products appear to have a molecular weight in excess of 150,000 as determined by gel filtration. Deceased.