Bile acid induction of cytokine expression by macrophages correlates with repression of hepatic cholesterol 7alpha-hydroxylase.
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In the studies reported herein, we show that two complementary experimental models: inbred strains of mice (i.e. C57BL/6 and C3H/HeJ), and a differentiated line of rat hepatoma cells (i.e. L35 cells), require the activation of cytokines by monocyte/macrophages to display bile acid negative feedback repression of cholesterol 7alpha-hydroxylase (CYP7A1). Feeding a bile acid-containing atherogenic diet for 3 weeks to C57BL/6 mice led to a 70% reduction in the expression of hepatic CYP7A1 mRNA, whereas no reduction was observed in C3H/HeJ mice. The strain-specific response to repression of CYP7A1 paralleled the activation of hepatic cytokine expression. Studies using cultured THP-1 monocyte/macrophages showed that the hydrophobic bile acid chenodeoxycholate, a well established potent repressor of CYP7A1, induced the expression of mRNAs encoding interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFalpha). In contrast, the hydrophilic bile acid ursodeoxycholate, which does not repress CYP7A1, did not induce cytokine mRNA expression by THP-1 cells. Chenodeoxycholate activation of cytokines by THP-1 cells was blocked by the peroxisome proliferator-activated receptor gamma agonist rosiglitazone. The expression of cytokines (e.g. IL-1 and TNFalpha) by THP-1 cells paralleled with the ability of these cells to produce conditioned medium that when added to rat L35 hepatoma cells, repressed CYP7A1. Moreover, rosiglitazone, which blocks cytokine activation by macrophages, also blocked the repression of CYP7A1 normally exhibited by C57BL/6 mice fed the bile acid-containing atherogenic diet. The combined data indicate that the activation of cytokines may mediate CYP7A1 repression caused by feeding mice an atherogenic diet containing bile acids. (+info)
Intracellular transport of bile acids.
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Bile acids originate from the liver and are transported via bile to the intestines where they perform an important role in the absorption of lipids and lipid-soluble nutrients. Most of the bile acids are reclaimed from the terminal ileum and returned to the liver via portal blood for reuse. The transport of bile acids is vectorial in both liver and intestinal cells, originating and terminating at opposite poles. Bile acids enter through the basolateral pole in liver cells, and through the apical pole in intestinal cells. During the past decade, much has been learned about the mechanisms by which bile acids enter and exit liver and intestinal cells. By contrast, the mechanisms by which bile acids are transported across cells remain poorly understood. The current body of evidence suggests that bile acids do not traverse the cell by vesicular transport. Although a carrier-mediated mechanism is a likely alternative, only a handful of intracellular proteins capable of binding bile acids have been described. The significance of these proteins in the intracellular transport of bile acids remains to be tested. (+info)
The nuclear matrix protein CDP represses hepatic transcription of the human cholesterol-7alpha hydroxylase gene.
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To date, the molecular mechanisms that govern hepatic-specific transcription of the human cholesterol 7alpha-hydroxylase (CYP7A1) gene are poorly understood. We recently reported that the region extending from -1888 to +46, which includes the promoter, is not capable of conferring expression to human CYP7A1 promoter lacZ transgenes in the livers of mice, but that expression is observed with transgenes containing the entire structural gene. To locate liver-specific elements in other segments of the human gene, DNase I hypersensitivity studies were performed with transcriptionally active, liver-derived HepG2 cells and with transcriptionally inactive HeLa cells. Three DNase I hypersensitivity sites were detected within the first intron of the human CYP7A1 gene, but only in HepG2 cells. Transient transfection experiments with HepG2 cells revealed a transcriptional repressor within intron 1. Five binding sites for the CAAT displacement protein (CDP) were detected within intron 1. Since CDP is a nuclear matrix protein, two methods were employed to localize nuclear matrix attachment sites within intron 1 of the human CYP7A1 gene. A matrix attachment site was found throughout the entirety of intron 1. Gel retardation experiments and cell transfection studies provided evidence for the repression mechanism. Repression is achieved by displacement by CDP of two hepatic activators, namely HNF-1alpha and C/EBPalpha, that bind to three different sites within intron 1. Additionally, CDP represses transactivation mediated by these two activators. (+info)
Oxysterol 7 alpha-hydroxylase activity by cholesterol 7 alpha-hydroxylase (CYP7A).
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A 7 alpha-hydroxylation is necessary for conversion of both cholesterol and 27-hydroxycholesterol into bile acids. According to current theories, cholesterol 7 alpha-hydroxylase (CYP7A) is responsible for the former and oxysterol 7 alpha-hydroxylase (CYP7B) for the latter reaction. CYP7A is believed to have a very high substrate specificity whereas CYP7B is active toward oxysterols, dehydroepiandrosterone, and pregnenolone. In the present study, 7 alpha-hydroxylation of various oxysterols in liver and kidney was investigated. Surprisingly, human cholesterol 7 alpha-hydroxylase, CYP7A, expressed as a recombinant in Escherichia coli and COS cells, was active toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. This enzyme has previously been thought to be specific for cholesterol and cholestanol. A partially purified and reconstituted cholesterol 7 alpha-hydroxylase enzyme fraction from pig liver showed 7 alpha-hydroxylase activity toward the same oxysterols as metabolized by expressed recombinant human and rat CYP7A. The 7 alpha-hydroxylase activity toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A. From the present results it may be concluded that CYP7A is able to function as an oxysterol 7 alpha-hydroxylase, in addition to the previously known human oxysterol 7 alpha-hydroxylase, CYP7B. These findings may have implications for oxysterol-mediated regulation of gene expression and for pathways of bile acid biosynthesis. A possible use of 20(S)-hydroxycholesterol as a marker substrate for CYP7A is proposed. (+info)
Biphasic effects of the natural estrogen 17beta-estradiol on hepatic cholesterol metabolism in intact female rats.
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The protective influence of estrogens in cardiovascular disease is believed to be partly due to beneficial effects on cholesterol metabolism. Much of the experimental data are based on models in which synthetic estrogens have been used in pharmacological doses, and therefore, the physiological role of estrogens in cholesterol metabolism is uncertain. To evaluate this important issue, we performed experiments in intact female rats with use of the natural estrogen 17beta-estradiol (E2) administered either subcutaneously or orally. After physiological doses of E2 (< or =0.04 mg. kg(-1). d(-1)) were administered, plasma levels of high density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-I were increased. In the liver, 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7alpha-hydroxylase activities were increased, as well as cholesterol 7alpha-hydroxylase mRNA levels. These effects were abolished during treatment with higher doses of E2, whereas apo A-I mRNA increased in a dose-dependent way. After treatment with pharmacological doses of E2 (> or =0.2 mg. kg(-1). d(-1)), the number of hepatic low density lipoprotein receptors increased and plasma cholesterol was reduced. These effects were similar after both oral and subcutaneous administration of E2. Our results show that the responses to E2 are biphasic: plasma HDL, apo A-I, and hepatic enzyme activities governing bile acid and cholesterol synthesis increased only at physiological doses of E2. At pharmacological doses of E2, hepatic low density lipoprotein receptors are stimulated and plasma cholesterol is reduced. Therefore, under physiological conditions, E2 exerts its major effects on hepatic cholesterol metabolism through mechanisms other than stimulation of low density lipoprotein receptor expression. (+info)
Effect of cyclosporine on HMG-CoA reductase, cholesterol 7alpha-hydroxylase, LDL receptor, HDL receptor, VLDL receptor, and lipoprotein lipase expressions.
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Long-term administration of cyclosporine (CsA) has been shown to cause hypercholesteremia, hypertriglyceridemia, and elevations of plasma low-density and very low-density lipoprotein (LDL and VLDL) levels in humans. This study was undertaken to explore the effects of CsA on expressions of the key lipid regulatory enzymes and receptors. Thus, hepatic expressions of cholesterol 7alpha-hydroxylase (the rate-limiting step in cholesterol conversion to bile acids), LDL receptor, and high-density lipoprotein (HDL) receptor proteins, as well as 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity were determined in rats treated with CsA (18 mg/kg/day) or placebo for 3 weeks. In addition, skeletal muscle and adipose tissue expressions of lipoprotein lipase and VLDL receptor were measured. Western blot analysis was used for all protein measurements using appropriate antibodies against the respective proteins. CsA-treated animals showed mild but significant elevations of plasma cholesterol and triglyceride concentrations. This was associated with a marked down-regulation of cholesterol 7alpha-hydroxylase in the liver and a severe reduction of lipoprotein lipase abundance in skeletal muscle and adipose tissue. However, hepatic LDL receptor and HDL receptor expressions and HMG-CoA reductase activity were not altered by CsA therapy. Likewise, skeletal muscle and adipose tissue VLDL receptor protein expressions were unaffected by CsA administration under the given condition. In conclusion, CsA administration for 3 weeks resulted in a significant reduction of hepatic cholesterol 7alpha-hydroxylase and marked down-regulation of skeletal muscle and adipose tissue lipoprotein lipase abundance in rats. The former abnormality can contribute to hypercholesterolemia by limiting cholesterol catabolism, whereas the latter may contribute to hypertriglyceridemia and VLDL accumulation by limiting triglyceride-rich lipoprotein clearance in CsA-treated animals. (+info)
Superiority of dietary safflower oil over olive oil in lowering serum cholesterol and increasing hepatic mRnas for the LDL receptor and cholesterol 7alpha-hydroxylase in exogenously hypercholesterolemic (exHC) rats.
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The exogenously hypercholesterolemic (ExHC) rat is a strain segregated from SD rats with a high response to dietary cholesterol. To understand the underlying mechanism(s) for this hypercholesterolemia, the interactive effects of dietary fatty acid and the susceptibility of rats to dietary cholesterol on the serum cholesterol concentration and hepatic mRNA abundance of the low-density lipoprotein (LDL) receptor, cholesterol 7alpha-hydroxylase (7alpha-hydroxylase) and 3-hydroxyl-3methylglutaryl (HMG) CoA reductase were examined. Both strains were fed on a diet supplemented with 10% each of olive, safflower or coconut oil with or without the addition of 1% cholesterol for one week. The ExHC rats fed on olive, safflower and coconut oil in combination with cholesterol respectively resulted in a 3.5-, 2.0- and 2.1-fold higher serum cholesterol concentration than that in the animals fed on the corresponding dietary fats without any supplementation of cholesterol (p < 0.01 by dietary cholesterol or type of fat). The dietary cholesterol dependent-elevation of serum cholesterol in the SD rats was less than 1.5-fold (p<0.01) and there was no dietary fat effect. The ExHC rats fed on the safflower oil-containing diet supplemented with cholesterol resulted in a higher mRNA abundance of the LDL receptor and 7alpha-hydroxylase than in the corresponding fat-fed rats without cholesterol (p<0.05). There was no dietary cholesterol-dependent change of mRNA abundance in either strain fed on olive or coconut oil, except for a decreased abundance of HMG CoA reductase mRNA in the olive oil-fed ExHC rats and coconut oil-fed Sprague-Dawley (SD) rats (p<0.05). These results indicate that the hepatic mRNA abundance of the LDL receptor and of 7alpha-hydroxylase depended on the dietary combination of cholesterol and a fatty acid and suggest that a linoleic acid-rich diet may alleviate exogenous hypercholesterolemia by activating the process involved in the hepatic uptake and biliary excretion of serum cholesterol. (+info)
Centripetal cholesterol flow from the extrahepatic organs through the liver is normal in mice with mutated Niemann-Pick type C protein (NPC1).
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Niemann-Pick type C (NPC) protein functions to move unesterified cholesterol from the lysosomal compartment to other intracellular sites for further metabolism and/or excretion. This cholesterol is brought into the cell through the coated-pit pathway and accumulates in the lysosomes when NPC protein is mutated. The present study quantitated the alternative uptake process that brings cholesterol into the cell through the scavenger receptor, class B, type I (SR-BI) pathway in animals with this mutation. In homozygous NPC mice, the tissues of the extrahepatic compartment accumulated an excess of 14 mg of cholesterol each day per kg body weight, and synthesis increased by a similar amount (to 111 mg/day per kg) to compensate for this functional loss of sterol through lysosomal sequestration. An amount of cholesterol (108 mg/day per kg) nearly equal to that synthesized in the extrahepatic compartment was carried through the circulation by high density lipoprotein (HDL) and taken up by the liver. The rate of hepatic cholesterol excretion from the NPC mice as fecal acidic (65 mg/day per kg) and neutral (85 mg/day per kg) sterols was elevated 61% above control values and was accounted for by the total amount of cholesterol brought to the liver in HDL and synthesized in the hepatocytes. These studies demonstrated that while cholesterol entering tissues of the NPC animals through the coated-pit pathway became sequestered in the lysosomal compartment and was metabolically inactive, cholesterol that was newly synthesized or that entered cells through the SR-BI pathway was metabolized and excreted normally. (+info)