Cholesterol and bile acid synthesis in Hep G2 cells. Metabolic effects of 26- and 7 α-hydroxycholesterol (original) (raw)
Related papers
26-Hydroxycholesterol: synthesis, metabolism, and biologic activities
Journal of Lipid Research, 1990
Cholest-5-ene-3&26-diol (26-hydroxycholesterol) is synthesized by a mitochondrial P-450 enzyme that appears to be widely distributed in tissues. Together with other (2-27 steroid intermediates it is transported to the liver and metabolized to bile acids. Although 26-hydroxycholestero1 is transported in plasma lipoproteins mostly as the fatty acid ester, neither its assembly and orientation within lipoproteins nor its mechanism of transport across the sinusoidal liver membrane is known. Cell culture studies indicate that 26-hydroxycholestero1 can inhibit both cholesterol synthesis and low density lipoprotein (LDL) receptor activity. Inhibition of DNA synthesis also occurs and may not be related to the reduction in HMG-CoA reductase activity. The relationship of these in vitro activities to the physiologic role(s) of 26-hydroxycholestero1 remains to be clarified. A clue to its biologic role is the knowledge that markedly decreased 26-hydroxylase activity appears to be the molecular basis of cerebrotendinous xanthomatosis, an inborn error of metabolism characterized by a significant decrease in 26-hydroxycholesterol and bile acid synthesis and an increase in cholesterol synthesis.
Journal of Clinical Investigation, 1978
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 synthesis 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. J. Clin. Invest.
Hepatology, 2003
The e&cts of newly synthesized cholesterol availability on bile acid synthesis are largely unknown, particularly in humans. The present study was aimed to study the changes induced on bile acid synthesis by simvastatin, a competitive inhibitor of hydroxymethyl glutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme of cholesterol synthesis, during pharmacologic interruption of the enterohepatic circulation. S i x patients with primary hypercholesterolemia were studied in basal conditions, after treatment with the bile acid binding resin cholestyramine alone (8-16 g/d for 6-8 weeks) and subsequently in combination with simvastatin (40 mg/d for 6-8 weeks). Cholesterol 7a-hydroxylation rate, a measure of total bile acid synthesis, was assayed in vim by tritium release analysis. Serum lathosterol levels were assayed by gas chromatographymass spectrometry as a measure of cholesterol synthesis. Serum total and low-density lipoprotein-cholesterol were reduced significantly after cholestyramine (by 26% and 30%, respectively) and during combined treatment (by 47% and 55%). 7a-Hydroxylation rates increased nearly 4-fold with cholestyramine alone; addition of simvastatin induced a significant decrease of hydroxylation rates (cholestyramine alone, 1,591 2 183 mg/d; plus simvastatin, 1,098 f 232 mg/dj mean 2 SEM; P < .05). Hydroxylation rates significantly correlated with serum lathosterou cholesterol ratio (T = 0.79, P < .05). In conclusion, in conditions of chronic stimulation bile acid synthesis may be affected by changes in newly synthesized cholesterol availability. The finding might relate to the degree of substrate saturation of microsomal cholesterol 7a-hydroxylase; alternatively, newly synthesized cholesterol might induce a stimulatory effkct on cholesterol 7a-hydroxylase transcription. (HEPATOLOGY 2003;38:939-946.) B ile acid production is a major mechanism whereby cholesterol is eliminated from the organism and, therefore, represents a crucial event in the maintenance of cholesterol homeostasis.1-3 Two metabolic path-Abbreviations: HMG-CoA, hydroxymethyl glutaryl-CoA; LDL, low-density lipoprotein; HDL, high-density lipoprotein.
Journal of Lipid Research, 1980
The present study was designed to obtain more definitive information in man on the metabolic pathways to chenodeoxycholic acid and to cholic acid via a pathway not involving an initial 7a-hydroxylation of cholesterol. Four bile fistula patients were administered consecutively two or more of the following 3H-labeled bile acid intermediates: 7a-hydroxycholestero1, 7a-hydroxy-4-cholesten-%one, 5P-cholestane 3a,7a,26-triol, 26-hydroxycholestero1, 7a1,26-dihydroxy-4-cholesten-3-one, and 5-cholestene-SP,-12a-diol. Both 7a-hydro~y[7/3-~H]cholesterol and 7a-hydro~y-4-[6/3-~H]cholesten-3-0ne were efficiently converted to bile acids and preferred chenodeoxycholic acid over cholic acid. The specific activity time curves indicated that a portion of cholic acid synthesis did not pass through 7a-hydroxycholesterol. [3H]26-Hydroxycholesterol and [3H]-5-cholestene 3P, 12a-diol, two potential intermediates of this bypass pathway to cholic acid, were poorly converted to primary bile acids (10 to 27%). The [3H]26-hydroxycholesterol preferred chenodeoxycholic over cholic acid by about 4 to 1. The [3H]5-cholestene 3/3,12a-diol formed cholic acid in low yield (10 to 20%). It is concluded that pathways to primary acids from cholesterol through 26-hydroxycholestero1 and 5-cholestene 3/3,12a-diol are probably of minor quantitative significance. A selective pathway to chenodeoxycholic acid via 26-hydroxylation of 7a-hydroxy-4-cholesten-3-one was also investigated. The 5P-cholestane 3a,7a,26-triol was converted in about equal amounts to cholic and chenodeoxycholic acids. The 7ahydroxy-4-cholesten-3-one was also efficiently converted to both bile acids but preferred chenodeoxycholic acid. The most efficient precursor of chenodeoxycholic acid was 7a,26-dihydroxy-4-cholesten-3-one, which was efficiently converted to primary bile acids; chenodeoxycholic acid was preferred over cholic acid by approximately 7 to 1. These findings suggest the presence of a major pathway to chenodeoxycholic acid via the 26-hydroxylation of 7a-hydroxy-4cholesten-3-one and intermediate formation of 7a,26dihydroxy-4-cholesten-3-one.-Swell L., J. Gustafsson, C. C. Schwa-, L. G. Halloran, H. Danielsson, and 2. R. Vlahcevic. An in vivo evaluation of the quantitative significance of several potential pathways to cholic and chenodeoxycholic acids from cholesterol in man. J. Lipid Res. 1980. 21: 455-466. Supplementary key words bile acids. metabolic pathways * 7a-hydroxylation * multiple pathways Cholic and chenodeoxycholic acids represent the major catabolic end products of cholesterol metabolism in man. These bile acids are synthesized from cholesterol in the liver cell by a series of reactions which involve epimerization of the 3P-hydroxy group, reduction of the A5 double bond to produce the 5P-configuration, introduction of a hydroxy groups at C, or C7 and Clz positions and oxidative degradation of the side chain by 3 carbons. Present concepts of the nature and sequence of enzymatic reactions involved in these transformations have been derived to a large extent from studies in the rat (1). Thc initial steps in the pathway are thought to be common for both primary bile acids and entail the microsomal 7ahydroxylation of cholesterol to form 7a-hydroxycholesterol which is then converted to 7a-hydroxy-4cholesten-3-one. The latter compound is considered to be the last intermediate common to the synthesis of cholic acid and chenodeoxycholic acid. This unsaturated ketone can then be reduced by soluble enzymes to 5P-cholestane 3a,7a-diol or 12a-hydroxylated in microsomes to form 7a, 12a-dihydroxy-4cholesten-3-one. This latter compound is then reduced to 5P-cholestane 3a,7a, 1 Pa-triol. According Abbreviations: GLC, gas-liquid chromatography; TLC, thinlayer chromatography.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1988
6-Azacholest-4-en-3/3-ol-7-one (azacholesterol) was shown to be a specific inhibitor of cholesterol 7a-hydroxylase. It inhibited cholesterol hydroxylation by a rat iiver microsomal preparation with non-competitive kinetics and a 1yi of 4 PM. No evidence was found for a time-blent inhibition of activity. Azachoiesterof did not inhibit acyl-CoA : cholesterol acyltransferase or 3-hy~xy-3-methylg~ut~~ coenzyme A reductase in rat liver microsomal preparations, or cholesterol esterification and synthesis in primary cultures of rat hepatocytes. The synthesis of bile acids was inhibited by azacholesterol in these cells in a dose-dependent way. When bile acid synthesis was inhibited by azacholesterol, newly-synthesised cholesterol from exogenous mevahmate was secreted by the hepatocyte cultures into the cell culture medium in severat-fofd excess over control incubations. No changes in the secretion of cholesteryi ester occurred in the presence of azacholesterol. This observation suggests that newly synthesised cholesterol that has entered the substrate pool for hydroxylation is no longer accessible to the substrate pool for esterification. This is further evidence for the compartmentation of cholesterol metabolism in the hepatocyte. introduction The liver presents one of the most complex patterns of intracellular cholesterol metabolism [I]. Because of its central role in cholesterol
Journal of Biological Chemistry
We measured hepatic cholesterol 7 alpha-hydroxylase activity, mass, and catalytic efficiency (activity/unit mass) in bile fistula rats infused intraduodenally with taurocholate and its 7 beta-hydroxy epimer, tauroursocholate, with or without mevalonolactone to supply newly synthesized cholesterol. Enzyme activity was measured by an isotope incorporation assay and enzyme mass by densitometric scanning of immunoblots using rabbit anti-rat liver cholesterol 7 alpha-hydroxylase antisera. Cholesterol 7 alpha-hydroxylase activity increased 6-fold, enzyme mass 34%, and catalytic efficiency 5-fold after interruption of the enterohepatic circulation for 48 h. When taurocholate was infused to the bile acid-depleted animals at a rate equivalent to the hepatic bile acid flux (27 mumol/100-g rat/h), cholesterol 7 alpha-hydroxylase activity and enzyme mass declined 60 and 61%, respectively. Tauroursocholate did not significantly decrease cholesterol 7 alpha-hydroxylase activity, mass and catalyti...
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1995
The effects of hyodeoxycholic (HDCA) and cu-hyocholic acids (a-HCA), on cholesterol, bile acid and lipoprotein metabolism, were studied in hamsters. The animals were fed a low cholesterol control diet supplemented with 0.1% HDCA or (Y-HCA for 3 weeks. In both treated groups, the LDL-cholesterol concentration was significantly lowered and was associated with a global hypocholesterolemic effect. Moreover, hepatic cholesterol ester storage was reduced and HMGCoA reductase activity was respectively enhanced 13.5-times and 7.7-times in HDCA and (Y-HCA groups compared to controls. In contrast, cholesterol 7cy-hydroxylase activity and LDL-receptor activity and mass were not modified. In bile, the cholesterol saturation index was increased 5-fold (HDCA group) and 2-fold (a-HCA group) as a consequence of an enlarged proportion of biliary cholesterol. The two 6-hydroxylated bile acids induced an enhanced fecal excretion of neutral sterols (HDCA group: 11.6-times, cr-HCA group: 3.2-times versus controls) which was consistent with a 59% decrease in intestinal cholesterol absorption in the HDCA group. The major effects due to bile acid treatments were a decrease in LDL-cholesterol concentration, a strong stimulation of hepatic cholesterol biosynthesis and an excessive loss of cholesterol in feces. These perturbations might be the result of the enrichment of bile with hydrophilic bile acids, leading to a limited return of endogenous cholesterol from the intestine to the liver.
Metabolism-clinical and Experimental, 1999
The regulation of the classic and alternative bile acid synthetic pathways by key hepatic enzyme activities (microsomal cholesterol 7oL-hydroxylase and mitochondrial sterol 27-hydroxylase, respectively) was examined in bile acid depletion and replacement and cholesterol-feeding experiments with rats, guinea pigs, and rabbits. The bile acid pool was depleted by creating a bile fistula (BF) and collecting bile for 2 to 5 days, and it was replaced by intraduodenal infusion of the major biliary bile acids (taurocholic acid [TCA], glycochenodeoxycholic acid [GCDCA], and glycocholic acid [GCA] in the rat, guinea pig, and rabbit, respectively) at rates equivalent to the measured hepatic flux of the bile acids. To study the effects of cholesterol, the animals were fed for 7 days on a basal diet with and without 2% cholesterol. Cholesterol 7~-hydroxylase and sterol 27-hydroxylase activities, measured by isotope incorporation assays, were related to bile acid output and composition and hepatic cholesterol concentrations. Intraduodenal infusion of bile acids increased the output of the tested bile acids, but did not significantly change hepatic cholesterol concentrations and had no effect on sterol 27-hydroxylase activity. Neither bile acid depletion nor replacement affected sterol 27-hydroxylase activity when three different substrates (cholesterol, 51~-cholestane-3~,7~-diol, and 51~-cholestane-3~,7~,12~-triol) were tested. In contrast, feeding 2% cholesterol increased hepatic cholesterol concentrations in rats, guinea pigs, and rabbits threefold, twofold, and eightfold, respectively, and increased hepatic mitochondrial stero127-hydroxylase activity (conversion of cholesterol to 27-hydroxycholesterol) in all three animal models. The stimulation and feedback inhibition of cholesterol 7~-hydroxylase activity by bile acid depletion and replacement were observed in all three animal models, whereas the effect of cholesterol feeding was species-dependent (cholesterol 7~-hydroxylase activity increased in the rat, did not change in the guinea pig, and was inhibited in the rabbit). Thus, in contrast to sterol 27-hydroxylase, which was upregulated by cholesterol but not affected by bile acid depletion and replacement in all three animal models, cholesterol 7~-hydroxylase activity was controlled consistently and inversely by the hepatic flux of bile acids, but was species-dependent in its response to a 1-week feeding with 2% cholesterol.
Journal of lipid research, 1983
After incubation in the presence of tritiated water, incorporation of tritium into cholesterol and into different bile acids was several-fold higher using hepatocytes of cholestyramine-fed rats than that found using hepatocytes of control rats. Labeling of the trihydroxylated cholic and beta-muricholic acids was markedly greater than that of dihydroxycholanoic acid. The total amount of label in all bile acids was 30% or less of that in free cholesterol, in both types of hepatocytes. In combination with the data on bile acids mass production we could calculate the average number (N(a)) of tritium atoms incorporated per molecule of newly-formed bile acid. The experimental values of N(a) for cholic and beta-muricholic acid were compared with values of N(n) or N(o), theoretically predicted if these bile acids were derived entirely from newly made or pre-existent cholesterol, respectively. It was deduced for hepatocytes of cholestyramine-fed rats that the bile acids produced in the first...