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Background: Transthyretin functions as a serum transport protein for retinol. Transthyretin has been found associated with amyloid diseases and it is an important nutrition indicator. For this study, we aimed to investigate the up and down stream molecular mechanisms of Transthyretin in cholesterol metabolism., Methods: We have recruited 237 fatty liver patients to evaluate the serum Transthyretin and its association with cholesterol and other clinical characteristics. And then Transthyretin was up and down regulated by plasmids to investigate its downstream mechanisms in vitro., Results: Linc00657 (NORAD) and miR-205-5p were further confirmed as upstream mechanisms to regulate Transthyretin. High level Transthyretin patients tended to have higher cholesterol, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) level than low level Transthyretin patients. Moreover, Transthyretin expressed higher in LO2 than that in QSG7701. Furthermore, Transthyretin negatively regulated CYP7A1, LXRα and ABCG 5/8 and positively regulated HMGCR and SREBP2. Linc00657 expressed lower in LO2 than that in QSG7701 and miR-205-5p expressed higher in LO2 than that in QSG7701. Furthermore, we found that linc00657 negatively regulated miR-205-5p and Transthyretin in vitro. And, up regulation of miR-205-5p in linc00657-LO2 cell line could reverse the inhibitory effects of linc00657 on Transthyretin., Conclusion: Transthyretin regulated cholesterol metabolism mainly through inhibiting LXRα-CYP7A1 and promoting SREBP2-HMGCR. And linc00657 could negatively regulate Transthyretin by inhibiting miR-205-5p, providing novel therapeutic targets for decreasing serum cholesterol level. Besides, Transthyretin could be a potential novel biomarker for predicting liver function along with AST and ALT., (Copyright © 2020. Published by Elsevier Inc.)

Aims: High-fat diet (HFD)-induced obesity resulting in cholesterol accumulation is one of the common pathogenic factors for lipids metabolic disorders. However, the potential mechanisms about cholesterol accumulation during obesity are still not clearly identified. Bile acids (BAs) as the natural ligands of farnesoid x receptor (Fxr) are demonstrated that can regulate the relevant enzymes and transporters at transcriptional level to determine the cholesterol homeostasis. Here, we explored the underlying mechanisms of hepatic cholesterol accumulation in HFD-induced obesity rats via the BAs-Fxr-enzymes/transporters signaling pathways., Materials and Methods: BAs and cholesterol levels as well as mRNA expressions of enzymes, transporters and nuclear receptors involving in cholesterol homeostasis in liver and ileum tissue were evaluated in 4-week HFD-induced obesity rats., Key Findings: HFD promoted BAs intestine passive absorption to increase the concentrations of BAs especially the chenodeoxycholic acids (CDCAs) in ileum of HFD-induced obesity rats. The increased CDCAs concentrations activated Fxr-Fgf15 pathway in ileum to result in the mRNA expression of Cyp7a1 in liver down-regulation, which inhibited cholesterol metabolizing into primary BAs to contribute to the cholesterol level increase in liver tissue in HFD-induced obesity rats., Significance: The hepatic cholesterol accumulation should be ascribed to the activation of ileum Fxr-Fgf15 pathway by the increased BAs passive absorption into ileal enterocytes under the condition of rats fed with HFD, which inhibited hepatic Cyp7a1 gene transcription to reduce metabolic elimination of cholesterol. Moreover, these findings are expected to provide a cue for the treatment of cholesterol metabolism disorders in obesity patient., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Polar cod is an abundant Arctic key species, inhabiting an ecosystem that is subjected to rapid climate change and increased petroleum related activities. Few studies have investigated biological effects of crude oil on lipid metabolism in this species, despite lipids being a crucial compound for Arctic species to adapt to the high seasonality in food abundance in their habitat. This study examines the effects of dietary crude oil exposure on transcription levels of genes related to lipid metabolism (peroxisome proliferator-activated receptors [ppar-α, ppar-γ], retinoic X receptor [rxr-β], palmitoyl-CoA oxidase [aox1], cytochrome P4507A1 [cyp7α1]), reproduction (vitellogenin [vtg-β], gonad aromatase [cyp19a1]) and biotransformation (cytochrome P4501A1 [cyp1a1], aryl hydrocarbon receptor [ahr2]). Exposure effects were also examined through plasma chemistry parameters. Additional fish were exposed to a PPAR-α agonist (WY-14,643) to investigate the role of PPAR-α in their lipid metabolism. The dose-dependent up-regulation of cyp1a1 reflected the activation of genes related to PAH biotransformation upon crude oil exposure. The crude oil exposure did not significantly alter the mRNA expression of genes involved in lipid homeostasis except for cyp7α1 transcription levels. Plasma levels of cholesterol and alanine transaminase showed significant alterations in fish exposed to crude oil at the end of the experiment. WY exposure induced a down-regulation of ppar-α, an effect contrary to studies performed on other fish species. In conclusion, this study showed clear effects of dietary crude oil exposure at environmentally relevant concentrations on xenobiotic biotransformation but revealed only weak alterations in the lipid metabolism of polar cod., (Copyright © 2018 Elsevier Inc. All rights reserved.)

To investigate the protective effect of glutamine (Gln) against obstructive cholestasis in association with farnesoid X receptor (FXR) activation, an obstructive cholestasis model was established in male Sprague-Dawley rats by bile duct ligation (BDL). Serum biomarkers and hematoxylin plus eosin staining were used to identify the degree of hepatic injury in the rats with obstructive cholestasis after Gln treatment. Immunohistochemistry, real-time PCR, Western blot, cultured primary rat hepatocytes with FXR knockdown, and dual-luciferase reporter assay were performed to elucidate the mechanisms underlying Gln hepatoprotection. We found that Gln treatment protected against obstructive cholestasis induced by BDL through reducing hepatocyte injury. Upregulation of the hepatic efflux transporters small heterodimer partner (Shp), bile salt export pump (Bsep), and multidrug resistance-associated protein 2 (Mrp2), and inhibition of the hepatic uptake transporter Na + /taurocholate cotransporting polypeptide (Ntcp) and the bile acid synthesis enzyme cholesterol 7α-hydroxylase (Cyp7a1) expression were observed in rats with BDL treated with Gln in vivo. Furthermore, the regulatory effect of Gln on Bsep and Mrp2 expression was abrogated after FXR knockdown in rat primary cultured hepatocytes. Luciferase assay HepG2 cells also illustrated FXR was a direct target for Gln treatment. In conclusion, the regulation of Bsep and Mrp2 expression mediated by FXR might be an important mechanism for Gln against obstructive cholestasis.

Background: A fascinating aspect of bile acid homeostasis is the coordination between bile acid uptake in intestine and hepatic bile acid synthesis. In response to bile acid uptake in enterocytes, farnesoid X receptor is activated and induces transcription of fibroblast growth factor (FGF)15 in mice, or FGF19 in humans. FGF15/19 is secreted into the enterohepatic circulation, and through activation of hepatic receptors, leads to repression of Cyp7a1, a rate-limiting enzyme for bile acid synthesis. Using a genetic approach, we identified a novel protein, Diet1, as a control point for FGF15/19 production., Key Messages: Mice with a Diet1-null mutation have reduced FGF15 secretion, causing impaired feedback repression of hepatic bile acid synthesis, and increased fecal bile acid excretion. As a result, Diet1-deficient mice constitutively convert cholesterol to bile acids and are resistant to diet-induced hypercholesterolemia and atherosclerosis. Diet1 affects FGF15/19 production at the posttranscriptional level, and the proteins appear to have overlapping subcellular localization in enterocytes. Diet1 appears to be a control point for the production of FGF15/19 in enterocytes, and thus a regulator of bile acid and lipid homeostasis. Studies to evaluate the role of common and rare DIET1 genetic variants in human health and disease are warranted., Conclusions: Further elucidation of the Diet1-FGF15/19 interaction will provide new insights into the intricate regulatory mechanisms underlying bile acid metabolism., (2015 S. Karger AG, Basel.)

Despite the efficacy in reducing acute rejection events in organ transplanted subjects, long term therapy with cyclosporine A is associated with increased atherosclerotic cardiovascular morbidity. We studied whether this drug affects the antiatherogenic process of the reverse cholesterol transport from macrophages in vivo. Cyclosporine A 50 mg/kg/d was administered to C57BL/6 mice by subcutaneous injection for 14 days. Macrophage reverse cholesterol transport was assessed by following [(3)H]-cholesterol mobilization from pre-labeled intraperitoneally injected macrophages, expressing or not apolipoprotein E, to plasma, liver and feces. The pharmacological treatment significantly reduced the amount of radioactive sterols in the feces, independently on the expression of apolipoprotein E in the macrophages injected into recipient mice and in absence of changes of plasma levels of high density lipoprotein-cholesterol. Gene expression analysis revealed that cyclosporine A inhibited the hepatic levels of cholesterol 7-alpha-hydroxylase, concomitantly with the increase in hepatic and intestinal expression of ATP Binding Cassette G5. However, the in vivo relevance of the last observation was challenged by the demonstration that mice treated or not with cyclosporine A showed the same levels of circulating beta-sitosterol. These results indicate that treatment of mice with cyclosporine A impaired the macrophage reverse cholesterol transport by reducing fecal sterol excretion, possibly through the inhibition of cholesterol 7-alpha-hydroxylase expression. The current observation may provide a potential mechanism for the high incidence of atherosclerotic coronary artery disease following the immunosuppressant therapy in organ transplanted recipients.

The COUP-TFII (chicken ovalbumin upstream promoter-transcription factor II) nuclear receptor, which is composed of a DNA-binding domain and a ligand-binding domain, exerts pleiotropic effects on development and cell differentiation by regulating the transcription of its target genes, including Cyp7a1 (cytochrome P450, family 7, subfamily a, polypeptide 1), which plays important roles in catabolism of cholesterol in the liver. Although multiple variants of COUP-TFII exist, their roles in the regulation of Cyp7a1 expression have not been elucidated. In the present study, we investigated the roles of COUP-TFII-V2 (variant 2), which lacks a DNA-binding domain, in the regulation of the transcriptional control of the Cyp7a1 gene by COUP-TFII in hepatocellular carcinoma cells. We found that COUP-TFII-V2 was significantly expressed in Huh7 cells, in which Cyp7a1 was not expressed. Furthermore, knockdown of COUP-TFII-V2 enhanced endogenous Cyp7a1 expression in Huh7 cells. Although COUP-TFII activates the Cyp7a1 promoter through direct binding to DNA, this activation was affected by COUP-TFII-V2, which physically interacted with COUP-TFII and inhibited its DNA-binding ability. Chromatin immunoprecipitation assays showed that COUP-TFII-V2 inhibited the binding of endogenous COUP-TFII to the intact Cyp7a1 promoter. The results of the present study suggest that COUP-TFII-V2 negatively regulates the function of COUP-TFII by inhibiting its binding to DNA to decrease Cyp7a1 expression.

Licorice (LE) has been commonly used in traditional Chinese medicine (TCM) for over 4000 years to reconcile various drugs and for hepatic disorders. Glycyrrhizin is the main bioactive component isolated from LE herbs. In the present study we examined the effects of glycyrrhizin on pregnane X receptor (PXR)-mediated CYP3A expression and its hepatoprotective activity. Treatment of HepG2 cells with glycyrrhizin resulted in marked increase in both CYP3A4 mRNA and protein levels. The transcriptional activation of the CYP3A4 gene through glycyrrhizin is PXR-dependent, as shown in transient transfection experiments. Glycyrrhizin activates the DNA-binding capacity of the PXR for the CYP3A4 element responding to xenobiotic signals, as measured by the electrophoretic-mobility shift assay (EMSA). These results indicate that the induction of the hepatic CYP3A4 by glycyrrhizin is mediated through the activation of PXR. The next aim of the current study was to determine whether the activation of PXR and induction of CYP3A by glycyrrhizin prevents hepatotoxicity during cholestasis as a mechanism of hepatoprotection. Mice were pretreated with glycyrrhizin prior to induction of intrahepatic cholestasis using lithocholic acid (LCA). Pre-treatment with glycyrrhizin, as well as the PXR activator pregnenolone 16α-carbontrile (PCN), prevents the increase in plasma ALT and AST activity, multifocal necrosis and prevents an increase in a level of serum LCA level in mice, as compared with the results in the mice treated with LCA alone. Activation of the PXR by glycyrrhizin results in induction of CYP3A11 (CYP3A4 for human) expression and inhibition of CYP7A1 through an increase in small heterodimer partner (SHP) expression. Glycyrrhizin regulates the expression of the gene mentioned above to prevent toxic accumulation of bile acids in the liver and it also protects mouse livers from the harmful effects of LCA. In conclusion, PXR-mediated effects on CYP3A and CYP7A may contribute to the hepatoprotective property of glycyrrhizin against LCA-induced liver injury., (Crown Copyright © 2012. Published by Elsevier Ireland Ltd. All rights reserved.)

Cholesterol 7α-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl(4))-induced fibrotic mouse liver, we demonstrated that the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Two sesquiterpenoids, atractylenolide II and III, were isolated and identified from Atractylodes macrocephala (Asteraceae) to be subsequently evaluated for their activity against farnesoid X receptor (FXR) and progesterone receptor (PR) by transient transfection reporter assays. These sesquiterpenoids did not exert significant agonistic effect but antagonized the activity of chenodeoxycholic acid (CDCA), an endogenous FXR agonist, for FXR driven SHP promoter transactivation. Additionally, they transactivated CYP7A1 gene promoter activity by antagonizing FXR. Apart from acting as a FXR antagonist, atractylenolide III also showed agonistic activity against PR. All these results demonstrated that atractylenolide II and III are the active components of Atractylodes macrocephala to exert specific pharmacologic effects., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background and Objective: Recent epidemiological studies have shown a correlation between periodontitis and hyperlipidemia. We have found high levels of oxidized low-density lipoprotein (OxLDL) in the gingival crevicular fluid of dental patients. In the present study, we tried to examine the possible role of OxLDL in periodontal inflammation in vitro., Material and Methods: Cells of the human gingival epithelial cell line Ca9-22 were cultured in media containing OxLDL, and the amounts of interleukin-8 (IL-8) and prostaglandin E(2) (PGE(2)) produced were measured using ELISAs., Results: Production of IL-8 by Ca9-22 cells was significantly increased when the cells were treated with OxLDL, but not with native LDL or acetylated LDL. Production of PGE(2) by Ca9-22 cells was enhanced by co-incubation with OxLDL and interleukin-1 beta (IL-1 beta). Scavenger receptor inhibitors, fucoidan and dextran sulfate, inhibited the OxLDL-induced IL-8 and PGE(2) production in the presence of IL-1 beta. The p(38) MAPK inhibitors SB203580 and SB202190 and the ERK inhibitor PD98059 inhibited the OxLDL-induced IL-8 production. Among oxidized lipids and chemically modified LDL, 7-ketocholesterol enhanced IL-8 production., Conclusion: This is the first report to show that OxLDL enhances IL-8 production in epithelial cells.

Vitamin D receptor (VDR) is activated by natural ligands, 1alpha, 25-dihydroxy-vitamin D(3) [1alpha,25(OH)(2)-D(3)] and lithocholic acid (LCA). Our previous study shows that VDR is expressed in human hepatocytes, and VDR ligands inhibit bile acid synthesis and transcription of the gene encoding cholesterol 7alpha-hydroxylase (CYP7A1). Primary human hepatocytes were used to study LCA and 1alpha,25(OH)(2)-D(3) activation of VDR signaling. Confocal immunofluorescent microscopy imaging and immunoblot analysis showed that LCA and 1alpha, 25(OH)(2)-D(3) induced intracellular translocation of VDR from the cytosol to the nucleus and also plasma membrane where VDR colocalized with caveolin-1. VDR ligands induced tyrosine phosphorylation of c-Src and VDR and their interaction. Inhibition of c-Src abrogated VDR ligand-dependent inhibition of CYP7A1 mRNA expression. Kinase assays showed that VDR ligands specifically activated the c-Raf/MEK1/2/extracellular signal-regulated kinase (ERK) 1/2 pathway, which stimulates serine phosphorylation of VDR and hepatocyte nuclear factor-4alpha, and their interaction. Mammalian two-hybrid assays showed a VDR ligand-dependent interaction of nuclear receptor corepressor-1 and silencing mediator of retinoid and thyroid with VDR/retinoid X receptor-alpha (RXRalpha). Chromatin immunoprecipitation assays revealed that an ERK1/2 inhibitor reversed VDR ligand-induced recruitment of VDR, RXRalpha, and corepressors to human CYP7A1 promoter. In conclusion, VDR ligands activate membrane VDR signaling to activate the MEK1/2/ERK1/2 pathway, which stimulates nuclear VDR/RXRalpha recruitment of corepressors to inhibit CYP7A1 gene transcription in human hepatocytes. This membrane VDR-signaling pathway may be activated by bile acids to inhibit bile acid synthesis as a rapid response to protect hepatocytes from cholestatic liver injury.

The conversion of cholesterol to bile acids is the major pathway for cholesterol catabolism. Bile acids are metabolic regulators of triglycerides and glucose metabolism in the liver. This study investigated the roles of FoxO1 in the regulation of cholesterol 7alpha-hydroxylase (CYP7A1) gene expression in primary human hepatocytes. Adenovirus-mediated expression of a phosphorylation defective and constitutively active form of FoxO1 (FoxO1-ADA) inhibited CYP7A1 mRNA expression and bile acid synthesis, while siRNA knockdown of FoxO1 resulted in a approximately 6-fold induction of CYP7A1 mRNA in human hepatocytes. Insulin caused rapid exclusion of FoxO1 from the nucleus and resulted in the induction of CYP7A1 mRNA expression, which was blocked by FoxO1-ADA. In high fat diet-fed mice, CYP7A1 mRNA expression was repressed and inversely correlated to increase hepatic FoxO1 mRNA expression and FoxO1 nuclear retention. In conclusion, our current study provides direct evidence that FoxO1 is a strong repressor of CYP7A1 gene expression and bile acid synthesis. Impaired regulation of FoxO1 may cause down-regulation of CYP7A1 gene expression and contribute to dyslipidemia in insulin resistance.

The role of farnesoid X receptor (FXR) in the development of atherosclerosis has been unclear. Here, LDL receptor (LDLR(-/-)) or apolipoprotein E (apoE(-/-)) female or male mice were fed a Western diet and treated with a potent synthetic FXR agonist, WAY-362450. Activation of FXR blocked diet-induced hypertriglyceridemia and elevations of non-HDL cholesterol and produced a near complete inhibition of aortic lesion formation. WAY-362450 also induced small heterodimer partner (SHP) expression and repressed cholesterol 7alpha-hydroxylase (CYP7A1) and sterol 12 alpha-hydroxylase (CYP8B1) expression. To determine if SHP was essential for these protective activities, LDLR(-/-)SHP(-/-) and apoE(-/-)SHP(-/-) mice were similarly treated with WAY-362450. Surprisingly, a notable sex difference was observed in these mice. In male LDLR(-/-)SHP(-/-) or apoE(-/-)SHP(-/-) mice, WAY-362450 still repressed CYP7A1 and CYP8B1 expression by 10-fold and still strongly reduced non-HDL cholesterol levels and aortic lesion area. In contrast, in the female LDLR(-/-)SHP(-/-) or apoE(-/-)SHP(-/-) mice, WAY-362450 only slightly repressed CYP7A1 and CYP8B1 expression and did not reduce non-HDL cholesterol or aortic lesion size. WAY-362450 inhibition of hypertriglyceridemia remained intact in LDLR(-/-) or apoE(-/-) mice lacking SHP of both sexes. These results suggest that activation of FXR protects against atherosclerosis in the mouse, and this protective effect correlates with repression of bile acid synthetic genes, with mechanistic differences between male and female mice.

Bioactive peptides with a variety of effects have been described from several nutritive proteins. They exhibit antimicrobial, blood-pressure lowering, antithrombotic, immunomodulatory, and cholesterol-modulating effects. In this study, we have examined whether peptides derived from food proteins might influence bile acid synthesis. A reporter gene cell line that carries a cholesterol 7alpha-hydroxylase promoter fragment fused to firefly luciferase ( cyp7a-luc) was used to screen for nutritive peptides affecting cyp7a expression, the enzyme catalyzing the rate-limiting step in bile acid synthesis. Proteolytic hydrolysates were prepared from soy protein and bovine casein with pepsin, trypsin, chymotrypsin, and elastase and size fractionated using ultrafiltration. Several bioactive hydrolysates could be identified that inhibited luciferase expression. Also, an activation of kinase (AKT, ERK, p38-MAPK) signaling could be observed. Selected hydrolysates were further fractionated by reversed-phase HPLC. Bioactive HPLC-fractions were obtained from casein but not from soy hydrolysates; however, activity could not be recovered in single peak fractions. Peptides in such fractions were identified by mass spectrometry. Five selected peptides from alpha S1-casein present in active fractions were synthesized, but none of these showed activity in the cyp7a-luc screening system. However, two of them activated MAP-kinase signaling similar to the hydrolysates, which suggests, that these peptides are involved in cyp7a regulation by the casein hydrolysates.

7-Ketocholesterol (7kCh) is a major oxysterol found associated with vascular diseases. Human microvascular endothelial cells (HMVECs) were cultured with different concentrations of 7kCh with and without inhibitors. Cell viabilities and caspase activities were assessed. 7kCh caused loss of cell viability in a dose-dependent manner. Caspases-8, -12, and -3/7 but not caspase-9 were activated by 7kCh treatment. The 7kCh-induced caspase-8 activity was blocked partially by pre-treatment with z-VAD-fmk and z-IETD-fmk, a caspase-8 inhibitor. However, pre-treatment with z-ATAD-fmk, a caspase-12 inhibitor, followed by 7kCh exposure lead to significantly increased caspase-8 activity. This suggests that caspase-8 and caspase-12 pathways have unique inhibition patterns and that caspase-12 is likely not upstream and feeding into caspase-8 but the pathways may function in parallel to each other. Caspase-3/7 activation was inhibited partially by low density lipoprotein (LDL), high density lipoprotein (HDL), z-VAD-fmk (pan-caspase inhibitor), and low doses (0.01 and 0.001 microM) of the cholesterol lowering drug, simvastatin. However, only LDL partially protected against 7kCh-induced loss of cell viability suggesting that caspase-independent pathways also contributed to the cell loss and that protection from oxysterol damage may require inhibition of multiple pathways. Moreover, our data suggest that oxysterols such as 7kCh can damage HMVECs cells in part via caspase-dependent apoptosis and may play a role in vascular and retinal diseases.

Purpose: To determine whether caspase or cathepsin pathways are activated in human retinal pigment epithelial cells (ARPE-19) after exposure to 7-ketocholesterol (7kCh)., Methods: ARPE-19 cells were exposed to 7kCh with or without z-VAD-fmk, a pan-caspase inhibitor. Caspase-3, -8, and -9 activities were measured by a fluorochrome inhibitor of caspase (FLICA) assay. Caspase-12 activity was detected by Western blotting. RT-PCR was performed for 18s, mortalin-2, cathepsins B, D, and L/V2., Results: At 24 hours, 7kCh-treated cultures had increased caspase-8 (P < 0.001) and caspase-3 (P < 0.001) activities compared with vehicle-treated cultures. 7kCh-induced caspase-3 activation was blocked by z-VAD-fmk (P < 0.001). Caspase-9 was not activated by 7kCh treatment (P > 0.05). Procaspase-12 was cleaved into its active form after treatment with 7kCh for 24 hours. At 6 hours, the RNA level for mortalin-2, a pro-survival gene, was upregulated. ARPE-19 cells did not express RNA for cathepsins B, D, or L/V2 under any conditions., Conclusions: In ARPE-19 cells, 7kCh-induced apoptosis uses the receptor-mediated caspase-8 pathway and the endoplasmic reticulum stress-induced caspase-12 pathway but not the mitochondrial caspase-9 pathway. The cathepsin pathways are not involved in 7kCh-induced cell death. These data demonstrate that 7kCh causes a loss of cell viability through caspase-dependent apoptosis and can act as an oxidative stressor leading to retinal pigment epithelial cell atrophy. Elucidating the specific apoptotic pathways involved may have therapeutic potential for AMD and other retinal diseases.

1. Previously, we found that Angelica keiskei extract (ethyl acetate extract from the yellow liquid of stems) elevated serum high-density lipoprotein (HDL) levels and reduced liver triglyceride content in stroke-prone spontaneously hypertensive rats (SHRSP). To identify the active substance in A. keiskei extract, we examined the effect of 4-hydroxyderricin, a characteristic chalcone isolated from the yellow liquid of stems, on blood pressure and lipid metabolism in SHRSP. 2. Six-week-old male SHRSP were fed diets containing 0.07% 4-hydroxyderricin for 7 weeks with free access to the diet and water. Elevation of systolic blood pressure was significantly suppressed after 7 weeks treatment. Serum very low-density lipoprotein (VLDL) levels were significantly reduced, without any effect on HDL levels, and were associated with a significant decrease in the serum concentration of free fatty acids. 3. In the liver, significant decreases in relative liver weight and triglyceride content were found after treatment with 4-hydroxyderricin for 7 weeks. 4. An investigation of hepatic mRNA expression of proteins involved in lipid metabolism indicated that a significant decrease in microsomal triglyceride transferprotein may be responsible for the decrease in serum VLDL levels and that significant decreases in adipocyte determination and differentiation factor 1 and fatty acid synthase may be responsible for the decrease in hepatic triglyceride content. 5. In conclusion, dietary 4-hydroxyderricin produces suppression of the elevation of systolic blood pressure, reduction of serum VLDL levels and a decrease in hepatic triglyceride content in SHRSP.

To study the effect of cholecystectomy on the regulation of classic and alternative bile acid syntheses, gallbladder-intact (n = 20) and cholecystectomized (n = 20) New Zealand White rabbits were fed either chow or chow with 2% cholesterol (3 g/day). After 10 days, bile fistulas were constructed in half of each rabbit group to recover and measure the bile acid pool and biliary bile acid flux. After cholesterol feeding, the bile acid pool size increased from 268 +/- 55 to 444 +/- 77 mg (P < 0.01) with a 2-fold rise in the biliary bile acid flux in intact rabbits but did not expand the bile acid pool (270 +/- 77 vs. 276 +/- 62 mg), nor did the biliary bile acid flux increase in cholecystectomized rabbits. Ileal apical sodium-dependent bile acid transporter protein increased 46% from 93 +/- 6 to 136 +/- 23 units/mg (P < 0.01) in the intact rabbits but did not change in cholecystectomized rabbits (104 +/- 14 vs. 99 +/- 19 units/mg) after cholesterol feeding. Cholesterol 7alpha-hydroxylase activity was inhibited 59% (P < 0.001) while cholesterol 27-hydroxylase activity rose 83% (P < 0.05) after cholesterol feeding in the intact rabbits but neither enzyme activity changed significantly in cholesterol-fed cholecystectomized rabbits. Fecal bile acid outputs reflecting bile acid synthesis increased significantly in the intact but not in the cholecystectomized rabbits fed cholesterol. Removal of the gallbladder prevented expansion of the bile acid pool after cholesterol feeding as seen in intact rabbits because ileal bile acid transport did not increase. As a result, cholesterol 7alpha-hydroxylase was not inhibited.

(24S)-Hydroxycholesterol is formed from cholesterol in the brain and is important for cholesterol homeostasis in this organ. Elimination of (24S)-hydroxycholesterol has been suggested to occur in the liver but little is known about the metabolism of this oxysterol. In the present investigation, we report formation of 7alpha, 24-dihydroxycholesterol in pig and human liver. 7alpha-hydroxylase activity toward both isomers of 24-hydroxycholesterol [(24S) and (24R)] was found in a partially purified and reconstituted cholesterol 7alpha-hydroxylase (CYP7A) enzyme fraction from pig liver microsomes. In contrast, a purified enzyme fraction of pig liver oxysterol 7alpha-hydroxylase with high activity toward 27-hydroxycholesterol did not show any detectable activity toward 24-hydroxycholesterol. 7alpha-Hydroxylation of 24-hydroxycholesterol was strongly inhibited by 7-oxocholesterol, a known inhibitor of CYP7A. Human CYP7A, recombinantly expressed in Escherichia coli and in simian COS cells, showed 7alpha-hydroxylase activity toward both cholesterol and the two isomers of 24-hydroxycholesterol, with a preference for the (24S)-isomer. Our results show that 24-hydroxycholesterol is metabolized by CYP7A, an enzyme previously considered to be specific for cholesterol and cholestanol and not active toward oxysterols. Because CYP7A is the rate-limiting enzyme in the major pathway of bile acid biosynthesis, the possibility is discussed that at least part of the 24-hydroxycholesterol is converted into 7alpha-hydroxylated bile acids by the enzymes involved in the normal biosynthesis of bile acids.

The effect of Ogi-Keishi-Gomotsu-To-Ka-Kojin (OKGK), a traditional Chinese herbal medicine (Kampo medicine), on cholesterol metabolism was studied in male Sprague-Dawley rats. Intake of OKGK at doses of 1.38 g/kg for 4 weeks significantly reduced total cholesterol levels in the serum and liver of hypercholesterolemia rats fed a cholesterol-enriched diet. OKGK suppressed cholesterol absorption through the intestine and stimulated excretion of cholesterol into feces as bile acids. Biochemical study indicated that OKGK treatment enhanced cholesterol 7alpha-hydroxylase activity the rate limiting enzyme of cholic acid synthesis, in the liver without any effect on the rate limiting enzyme of cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Further, cholesterol-enriched diet containing cholic acid suppressed cholesterol 7alpha-hydroxylase activity, whereas OKGK administration reversed the suppression. In conclusion, these results supported the idea that OKGK may be an effective agent for treatment of patients with hypercholesterolemia.

Background & Aims: Cholesterol degradation to bile acids occurs via "classic" or "alternative" bile acid biosynthetic pathways. The aim of this study was to assess the contributions of these two pathways to total bile acid synthesis in vivo., Methods: Rats with biliary fistulas were infused with squalestatin for 24 and 48 hours; specific activities of cholesterol 7 alpha-hydroxylase (C7 alpha H) and sterol 27-hydroxylase (S27H) and rates of bile acid synthesis were determined., Results: Continuous squalestatin infusion (15 micrograms/h) decreased C7 alpha H specific activities to 4% and 12% of paired biliary fistula controls at 24 and 48 hours, respectively (P < 0.05) without any changes in S27H specific activities (82% and 95% of controls). At 24 hours, bile acid synthesis decreased to 43% (P < 0.05) but returned to 87% at 48 hours (P = NS). Cholic acid synthesis decreased at 24 hours but returned to control levels at 48 hours. Similar changes in C7 alpha H, S27H, and bile acid synthesis were observed in primary rat hepatocytes after addition of squalestatin (1.0 mumol/L)., Conclusions: In the face of persistent suppression of C7 alpha H and the classic pathway, an alternative pathway becomes a main pathway of bile acid synthesis capable of generating cholic and chenodeoxycholic acids. The observed induction of bile acid synthesis via an alternative pathway or pathways represents an important mechanism for maintenance of cholesterol homeostasis in the rat.

Background & Aims: Cholesterol feeding unexpectedly inhibits cholesterol 7 alpha-hydroxylase in rabbits. The aim of this study was to explore the mechanism., Methods: Twenty male New Zealand white rabbits were fed regular chow with and without 2% cholesterol for 10 days followed by 7 days of bile drainage. The activities of hepatic cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase that control bile acid synthesis in classic and alternative pathways were related to the size and composition of bile acid pool., Results: After feeding cholesterol, plasma and hepatic cholesterol concentrations increased, the bile acid pool doubled (from 254 +/- 44 to 533 +/- 51 mg; P < 0.001), cholesterol 7 alpha-hydroxylase activity decreased 68% (P < 0.01), but sterol 27-hydroxylase activity increased 66% (P < 0.05) with increased cholic acid synthesis (P < 0.01). Bile drainage in the cholesterol-fed rabbits depleted the bile acid pool and stimulated down-regulated cholesterol 7 alpha-hydroxylase activity 11.4-fold (P < 0.001), although hepatic cholesterol remained elevated. Hepatic sterol 27-hydroxylase activity was unaffected., Conclusions: Feeding cholesterol increased hepatic cholesterol and stimulated sterol 27-hydroxylase and alternative bile acid synthesis, which expanded the bile acid pool and inhibited cholesterol 7 alpha-hydroxylase in rabbits. In distinction, hepatic sterol 27-hydroxylase was insensitive to changes in the bile acid pool.

Consumption of boiled coffee raises serum cholesterol levels in humans. The diterpenes cafestol and kahweol in boiled coffee have been found to be responsible for the increase. To investigate the biochemical background of this effect, we studied the effects of cafestol and a mixture of cafestol/kahweol/isokahweol (48:47:5 w/w) on bile acid synthesis and cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase in cultured rat hepatocytes. Dose-dependent decreases of bile acid mass production and cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase activity were found, showing a maximal reduction of -91%, -79%, and -49% respectively, at a concentration of 20 micrograms/mL cafestol. The decrease in 7 alpha-hydroxylase and 27-hydroxylase activity paralleled well the suppression of the respective mRNAs, being -79% and -77%, and -49% and -46%, respectively, at 20 micrograms/mL cafestol. Run-on data showed a reduction in 7 alpha-hydroxylase and 27-hydroxylase gene transcriptional activity after incubation with cafestol. The mixture of cafestol/kahweol/isokahweol was less potent in suppression of bile acid synthesis and cholesterol 7 alpha-hydroxylase. Cafestol (20 micrograms/mL) had no effect on lithocholic acid 6 beta-hydroxylase mRNA, another enzyme involved in bile acid synthesis. LDL-receptor, HMG-CoA reductase, and HMG-CoA synthase mRNAs were significantly decreased by cafestol (-18%, -20%, and -43%, respectively). We conclude that cafestol suppresses bile acid synthesis by downregulation of cholesterol 7 alpha-hydroxylase and of, to a lesser extent, sterol 27-hydroxylase in cultured rat hepatocytes, whereas kahweol and isokahweol are less active. We suggest that suppression of bile acid synthesis may provide an explanation for the cholesterol-raising effect of cafestol in humans.

The effect of the sulfur-substituted fatty acid analogue 1,10 bis(carboxymethylthio)decane, also known as 3-thiadicarboxylic acid, on puromycin aminonucleoside-induced nephrotic hyperlipidemia was studied in rats. Treatment with 3-thiadicarboxylic acid (250 mg/kg) for 5 days reduced plasma levels of triglycerides from 5.8 to 2.7 mmol/L and cholesterol from 11.0 to 7.7 mmol/L. This was accounted for by decreases in very-low-density lipoprotein triglycerides, very-low-density lipoprotein cholesterol, and low-density lipoprotein cholesterol, without any major changes in the composition of plasma lipoproteins. The activities of two enzymes involved in fatty acid synthesis (ATP:citrate lyase and fatty acid synthetase) were inhibited by 3-thiadicarboxylic acid treatment, whereas acetyl-coenzyme A carboxylase activity was unchanged. In contrast, treatment with the sulfur-substituted fatty acid analogue induced the peroxisomal beta-oxidation of fatty acids ninefold and the mitochondrial beta-oxidation by 54% to 73%, depending on the substrate used. This was accompanied by a 26% reduction in hepatic triglyceride secretion rate. The hepatic phosphatidate phosphohydrolase activity was unchanged. 3-Thiadicarboxylic acid treatment suppressed the activity of the rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, by 58%, whereas hepatic LDL receptor expression was unaltered. The activities of lipoprotein lipase and hepatic lipase were unchanged by treatment. These results demonstrated that treatment with 3-thiadicarboxylic acid ameliorates hyperlipidemia in experimental nephrosis primarily by decreasing the overproduction of very-low-density lipoprotein present. The data also indicate that hepatic very-low-density lipoprotein synthesis and secretion is strongly influenced by the availability of the fatty acid substrate under the same hyperlipidemic conditions.

Rats of the Sprague-Dawley strain were infused intravenously with a fat emulsion (Intralipid, trademark of Kabi Pharmacia, Uppsala, Sweden) containing 7-oxocholesterol. This resulted in an increased cholesterol 7 alpha-hydroxylase activity in liver microsomes as compared to controls and was accompanied by increased levels of cholesterol 7 alpha-hydroxylase mRNA and microsomal cholesterol 7 alpha-hydroxylase protein. Rats were also fed a cholestyramine-supplemented diet and infused with 7-oxocholesterol. These animals excreted about half as much bile acids in faeces as cholestyramine-fed controls. Addition of 7-oxocholesterol to liver microsomes from normal rats in amounts corresponding to those present in microsomes from 7-oxocholesterol-treated rats inhibited the cholesterol 7 alpha-hydroxylase activity by about 75%. Cholesterol induced a type-I binding spectrum when added to a purified bacterial-expressed cholesterol 7 alpha-hydroxylase (P-450c7 delta 2-24). 7-Oxocholesterol competitively inhibited the cholesterol binding spectrum, while 7 beta-hydroxycholesterol did not interfere with binding of cholesterol to the enzyme. It is concluded that treatment with the competitive inhibitor 7-oxocholesterol leads to a reduced bile acid biosynthesis and, as a consequence of reduced bile acid inhibition, a compensatory increase in cholesterol 7 alpha-hydroxylase synthesis. The high enzyme activity measured in microsomal preparations from 7-oxocholesterol-treated rats may be due to a continuous conversion of 7-oxocholesterol into less inhibitory metabolites, e.g. 7 beta-hydroxycholesterol. The latter compound was found in high concentrations in liver microsomes from rats treated with 7-oxocholesterol. The physiological importance of these results is discussed in relation to the previous findings that 7-oxocholesterol is accumulated in liver after cholesterol feeding and that 7-oxocholesterol is formed from cholesterol during lipid peroxidation.

Cultured rat hepatocytes continually synthesize cholesterol form radiolabeled acetate during a 24 h incubation period and export it, presumably as VLDL (very low density lipoprotein) to the culture medium. Mevastatin inhibits cholesterol biosynthesis by 90%. Incubation of the cultures with water-soluble extracts of garlic powder (Kwai, Sapec) diminish cholesterol biosynthesis (20-25%) as well as its export into the medium (30-35%). The IC50-value is 90 micrograms/ml. Between about 0.25 and 10 mg/ml the average maximal inhibition amounts to about 23%. Cytotoxicity of the extracts is apparent at concentrations above 125 mg/ml only. Pure alliin alone, or after incubation with alliinase (conversion to allicin) in concentrations corresponding to its content in the extracts does not exert any inhibition. Replacement of 14C-acetate by 14C-mevalonate omits the inhibitory effect. The activity of HMGCoA (hydroxymethylglutaryl-CoA) reductase is significantly reduced by garlic extracts at 50 micrograms/ml. At higher concentrations fatty acid synthetase, cholesterol 7 alpha-hydroxylase and cholesterol acyltransferase are slightly inhibited. Fatty acid synthetase is the only one of these enzymes which is inhibited by alliin at very high concentrations. These results demonstrate that water-soluble garlic extracts diminish hepatic cholesterol biosynthesis, thus contributing to the reduction of blood cholesterol. The main target site seems to be HMGCoA-reductase. The actual active principle(s) is still unknown. Alliin, however, does not seem to be of major significance.

The oxysterol 25-hydroxycholesterol acts both as a regulatory sterol determining the expression of genes governed by sterol regulatory elements and as a substrate for 7-alpha-hydroxylase, the first and rate-limiting enzyme in the bile acid synthetic pathway. Most wild-type nonhepatic cells are killed by the cytotoxic action of 25-hydroxycholesterol. In contrast, liver cells, which express 7-alpha-hydroxylase activity, are resistant to killing by 25-hydroxycholesterol. We examined the possibility that selection for resistance to 25-hydroxycholesterol might lead to the derivation of a cell line expressing 7-alpha-hydroxylase. A rat hepatoma cell line (7-alpha-hydroxylase minus) was transfected with human DNA and screened for resistance to 25-hydroxycholesterol. Although parental hepatoma cells were all killed within a week, a 25-hydroxycholesterol-resistant cell line (L35 cells) which showed stable expression of 7-alpha-hydroxylase activity and mRNA was obtained. These cells exhibited normal inhibition of cholesterol biosynthesis by 25-hydroxycholesterol. Blocking 7-alpha-hydroxylase activity with ketoconazole also blocked the resistance of L35 cells to 25-hydroxycholesterol. Isolation of microsomes from these cells showed levels of 7-alpha-hydroxylase activity (22.9 pmol/min/mg of protein) that were comparable to the activity (33.2 pmol/min/mg) of microsomes isolated from the livers of rats killed during the high point of the diurnal cycle. Parental cells had no detectable activity. These data show a new complementation group for 25-hydroxycholesterol resistance: expression of 7-alpha-hydroxylase. Dexamethasone increased both the activity and the cellular content of mRNA coding for 7-alpha-hydroxylase. Since dactinomycin blocked the ability of dexamethasone to induce mRNA, active transcription is required. Southern analysis of genomic DNA showed that L35 cells contain the rat (endogenous) gene but not the human gene. Furthermore, the RNA expressed by L35 cells is similar in size to rat RNA and is distinct from the human form of 7-alpha-hydroxylase. The combined data indicate that L35 cells are resistant to 25-hydroxycholesterol because they express 7-alpha-hydroxylase. The mechanism responsible involves activation of the endogenous (silent) gene of the parental rat hepatoma cell.

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 catalytic efficiency. The administration of mevalonolactone, which is converted to cholesterol, modestly increased cholesterol 7 alpha-hydroxylase activity and enzyme mass in the bile acid-depleted rats. However, when taurocholate was infused together with mevalonolactone, cholesterol 7 alpha-hydroxylase activity and catalytic efficiency were markedly depressed while enzyme mass did not change as compared with bile acid-depleted rats. These results show that (a) hepatic bile acid depletion increases bile acid synthesis mainly by activating cholesterol 7 alpha-hydroxylase with only a small rise in enzyme mass, (b) replacement with taurocholate for 24 h decreases both cholesterol 7 alpha-hydroxylase activity and mass proportionally, (c) when cholesterol is available (mevalonolactone supplementation), the infusion of taurocholate results in the formation of a catalytically less active cholesterol 7 alpha-hydroxylase, and (d) tauroursocholate, the 7 beta-hydroxy epimer of taurocholate, does not inhibit cholesterol 7 alpha-hydroxylase. Thus, bile acid synthesis is modulated by the catalytic efficiency and mass of cholesterol 7 alpha-hydroxylase. The enterohepatic flux of 7 alpha-hydroxylated bile acids and the formation of hepatic cholesterol apparently control cholesterol 7 alpha-hydroxylase by different mechanisms.

The effect of ursodeoxycholic acid analogues bearing modifications at the side-chain moiety of the molecule was tested on cholesterol 7 alpha-hydroxylase and HMG-CoA reductase in rat liver microsomes. The compounds included 23 R,S mixture and the single isomers 23R and 23S of 23 methylursodeoxycholic acid (23-methyl UDCA), the isomeric mixture (cis + trans) of 3 alpha,7 beta-dihydroxy-20,22-methylen-5 beta-cholan-23-oic acid (norcypro-UDCA) and the corresponding single isomers. Each steroid was added to liver microsomes as the sodium salt, at concentrations ranging from 25 to 200 microM. Isomers 23R and 23S of 23-methyl-UDCA inhibited cholesterol 7 alpha-hydroxylase in a concentration-dependent manner. The inhibitory capacity was similar for the two isomers. The extent of inhibition of the analogues was greater than that of the parent compound UDCA. Shortening of the side-chain in norcypro-UDCA resulted in a partial loss of the inhibitory effect, as compared to cypro-UDCA (3 alpha,7 beta-dihydroxy-22,23-methylen-5 beta-cholan-24-oic acid). None of these bile acid derivatives affected the activity of the enzyme HMG-CoA reductase.

Feedback regulation of bile acid synthesis by its end products was studied in cultured hepatocytes of young weaned pigs. We previously showed that conversion of exogenous [14C] cholesterol into bile acids was suppressed by addition of bile acids to the culture medium. In the present study, the effects of bile acids on bile acid mass production and cholesterol 7 alpha-hydroxylase activity were examined. Mass production of bile acids was strongly inhibited by addition of taurocholic acid (50 and 100 mumol/L) to the culture medium. The inhibitory action was exerted specifically on activity of cholesterol 7 alpha-hydroxylase because conversion of [14C] 7 alpha-hydroxycholesterol to bile acids by pig hepatocytes was not affected. Suppression of cholesterol 7 alpha-hydroxylase activity after incubation of the hepatocytes with taurocholic acid was concentration- and time-dependent. Maximum suppression (-80%) was achieved after a 20 to 30 hr incubation of hepatocytes with 100 mumol/L of this bile acid. Decline of enzyme activity caused by 100 mumol/L taurocholic acid followed first-order kinetics with a half-life of 10 hr. Taurocholic acid had no direct effect on cholesterol 7 alpha-hydroxylase activity in homogenates of hepatocytes as assessed by addition of the bile acid to the assay mixture. The effects of several other bile acids in a concentration of 100 mumol/L on cholesterol 7 alpha-hydroxylase activity were examined in 48 hr incubations. Glycochenodeoxycholic and glycohyodeoxycholic acids, which are the major bile acids in pig bile, their unconjugated forms and also deoxycholic and cholic acid pronouncedly inhibited activity of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Different concentrations of polar fractions, methanol-soluble (MESF), or water-soluble (WASF), of 1-8% equivalent to fresh garlic paste were added to yellow corn-soybean based diets and fed to 5-week-old male broiler chickens for 3 weeks to measure the inhibition of hepatic beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase, cholesterol 7 alpha-hydroxylase (7 alpha-hydroxy) and fatty acid synthetase (FAS). Dose-related decreases in the activities of these enzymes were obtained. Decreases in serum total cholesterol and in low density lipoprotein (LDL) levels were also observed. There was no effect on the level of cholesterol in high density lipoprotein (HDL). The most effective dose for these decreases was found 0.54% (MESF) and 1.2% (WASF) equivalent to 6% of the fresh garlic. The inhibition of HMG-CoA reductase and FAS by 25-300 micrograms of MESF or WASF for 15 min was tested in vitro, in male and female chicken hepatocytes. Inhibitions of activity were dose-dependent and the degree of inhibition increased with duration of incubation (150 micrograms of MESF or WASF 5 to 60 min). Dietary supplementation of odorless WASF of garlic was found to be very effective in lowering the total and LDL cholesterol levels compared to control chickens.

Treatment of neonatal guinea pigs with cholestyramine persistently increases the activity of hepatic cholesterol 7 alpha-hydroxylase (CH-7 alpha), the rate-limiting enzyme of bile acid biosynthesis. In this study, we examined whether CH-7 alpha activity could be persistently inhibited by manipulations (chenodeoxycholic acid feeding) during neonatal life. Male Hartley guinea pigs (2-3 days old) were fed 0.25% chenodeoxycholic acid (CDCA)-supplemented diet for 10 days or 6 weeks. Feeding CDCA for 10 days increased plasma cholesterol [controls (C), 25 +/- 3 vs. CDCA, 34 +/- 2 mg/dl]. Bile acid pool in animals fed CDCA for 10 days was nearly fivefold greater than controls (C, 5.8 +/- 0.3 vs. CDCA, 29.1 +/- 0.9 mg/100 g body weight), whereas CH-7 alpha activity was almost completely inhibited [C, 1.83 +/- 0.4 vs. CDCA, 0.02 +/- 0.02 pmol/(mg . minute)]. Four weeks after stopping CDCA feeding, CH-7 alpha was greater in the CDCA-fed animals (C, 0.47 +/- 0.03 vs. CDCA, 0.67 +/- 0.05). This was associated with a greater bile acid pool in these animals (C, 3.0 +/- 0.2 vs. CDCA, 5.8 +/- 0.4). We conclude that CDCA fed to neonatal guinea pigs inhibits CH-7 alpha activity. This inhibition is not permanent, however, since CH-7 alpha activity increases following withdrawal from chronic CDCA feeding.

Effects of carbon monoxide, nitrogen, ferricytochrome c and p-hydroxymercuribenzoate were studied on cholesterol 7 alpha-hydroxylase activity of swine hepatic microsomes. The results suggest that a microsomal electron transport system is involved in hepatic microsomal cholesterol 7 alpha-hydroxylation in swine. Cholesterol 7 alpha-hydroxylation is inhibited by superoxide dismutase in the standard assay system containing a NADPH generating system. Superoxide dismutase also inhibited cholesterol 7 alpha-hydroxylation in the system where superoxides were generated by enzymatic or nonenzymatic means in the absence of NADPH-generating system. The current study suggests that superoxide anion may be an important factor in the cholesterol 7 alpha-hydroxylation of swine.

The effect of the catatoxic steroid, 3beta-hydroxy-20-oxo-5-pregnene-16alpha-carbonitrile [pregnenolone-16alpha-carbonitrile (PCN)] on hepatic microsomal cholesterol-7alpha-hydroxylase, the probable rate-limiting enzyme of bile acide biosynthesis, has been studied. Short term administration (3 days) of PCN in the diet of rats resulted in a significant decrease in the liver microsomal cholesterol-7alpha-hydroxylase activity, in contrast to a marked stimulation of microsomal cytochrome P-450 and ethylmorphine demethylase activity. PCN significantly depressed the cholesterol-7alpha-hydroxylase activity in the livers of rats with elevated levels of the enzyme produced by cholestyramine feeding. The results indicate the presence of separate control mechanisms in the regualtion of bile acid synthesis and drug metabolism.

6-Azacholest-4-en-3 beta-ol-7-one (azacholesterol) was shown to be a specific inhibitor of cholesterol 7 alpha-hydroxylase. It inhibited cholesterol hydroxylation by a rat liver microsomal preparation with non-competitive kinetics and a Ki of 4 microM. No evidence was found for a time-dependent inhibition of activity. Azacholesterol did not inhibit acyl-CoA: cholesterol acyltransferase or 3-hydroxy-3-methylglutaryl 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-synthesized cholesterol from exogenous mevalonate was secreted by the hepatocyte cultures into the cell culture medium in several-fold excess over control incubations. No changes in the secretion of cholesteryl 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.

In cultured hepatocytes conversion of [4-14C]cholesterol into bile acids was dose dependently reduced by the antimycotic drug ketoconazole, giving half-maximal inhibition at 10 microM ketoconazole in rat hepatocytes and at 1 microM in human hepatocytes. No change was observed in the ratio of produced cholic, beta-muricholic, and chenodeoxycholic acid with increasing amounts of the drug. Conversion of [4-14C]7 alpha-hydroxycholesterol, an intermediate of bile acid pathway, to bile acids was not affected by ketoconazole. These results together with kinetic studies with rat liver microsomes, demonstrating noncompetitive inhibition (Ki = 0.4 microM), indicate that cholesterol 7 alpha-hydroxylase is the main site of inhibition. In bile-diverted rats a single dose of ketoconazole (50 mg/kg) dramatically impaired bile flow and biliary bile acid output (92% inhibition). A similar blockade was observed using [4-14C]cholesterol as precursor for bile acid synthesis. Therefore, treatment of patients with this drug may inhibit bile acid synthesis, resulting in a reduction of the bile acid pool size after long-term ketoconazole therapy.

A sensitive and precise method is described to assay cholesterol 7 alpha-hydroxylase activity in homogenates of rat hepatocytes cultured in monolayers for up to 76 h. The assay is based on measurement of the amount of radioactive cholesterol converted into 7 alpha-[14C]-hydroxycholesterol. Since no subcellular fractionation was applied to measure enzyme activity, this method is rapid and can be performed with cell protein, corresponding to as little as 1 to 2 million hepatocytes. Optimal assay conditions were determined and the reproducibility of this cholesterol 7 alpha-hydroxylase determination was established. Exogenous cholesterol (105 microM), solubilized in Tween 80, was added to saturate the enzyme, giving an apparent Km of 56 microM. Under these conditions, 70% of the cholesterol present in the homogenates is directly accessible to the cholesterol 7 alpha-hydroxylase. The detection limit of the assay was found to be about 10 pmol per incubation. A time course of the cholesterol 7 alpha-hydroxylase activity in cultured hepatocytes revealed that after an initial loss of approximately 60% of the activity as compared with 287 pmol/h/mg for freshly isolated cells, the enzyme activity was increased to the initial level in hepatocytes cultured for 52 h. This result and the finding that the cholesterol 7 alpha-hydroxylase activity was diminished by 94% after a 24-h incubation with 5 microM cycloheximide suggest that the enzyme activity is associated with de novo protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vitro effects of prostaglandins E1 and F1alpha on the activity of cholesteryl ester synthetase and cholesteryl ester hydrolase activities of the pigeon aorta were examined. It was found that prostaglandin E1 markedly inhibited the cholesteryl ester hydrolase activity in the supernatant fraction and slightly inhibited the cholesteryl ester synthetase activity. Prostaglandin F1alpha, however, modestly stimulated the cholesteryl ester hydrolase activity both in the microsomal and in the supernatant fraction of the aorta. These observations strongly warrant further studies on the role of prostaglandins in atherogenesis.

Cytochrome P-450Ch7 alpha (cholesterol 7 alpha-hydroxylase) catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids in mammalian liver. The properties of this cytochrome P-450 (P-450) form were studied in rat hepatic microsomal preparations in comparison to those of several well characterized constitutive and xenobiotic-inducible rat hepatic P-450s. Administration of the bile acid-sequestering resin cholestyramine [4% (w/w) in the diet] to male or female rats maintained on a reverse light cycle led to a 10- to 15-fold induction of P-450Ch7 alpha activity relative to untreated, standard light cycle controls. By contrast, the levels of four hepatic steroid hormone hydroxylating P-450 enzymes, designated 2a, 2c, 3, and PB-4 [Waxman, D.J. (1984) J. Biol. Chem. 259, 15481-15490], were not significantly affected by cholestyramine treatment. Antibody inhibition experiments established that P-450Ch7 alpha is immunochemically distinct from nine other rat hepatic P-450s, including P-450 3, a highly regio- and stereoselective steroid hormone 7 alpha-hydroxylase. P-450Ch7 alpha was shown to be selectively inactivated by micromolar concentrations of the disulfide-containing reagents disulfiram (Antabuse) and 2,2'-dithiopyridine. This inactivation was readily reversed upon incubation with 2-mercaptoethylamine, suggesting the presence of a highly reactive thiol group at the active site of P-450Ch7 alpha. These findings demonstrate that P-450Ch7 alpha corresponds to a unique P-450 enzyme exhibiting inductive, biochemical, immunochemical, and regulatory properties distinct from those of nine well-characterized rat hepatic P-450 forms.

The effect of chenodeoxycholic (CDCA), ursodeoxycholic (UDCA), tauroursodeoxycholic (TUDCA), cholic (CA), ursocholic (UCA) acids, analogues of CDCA and UDCA with a cyclopropyl ring at C22, C23 (cypro-CDCA and cypro-UDCA) and 23-methylursodeoxycholic acid (MUDCA) on cholesterol 7 alpha-hydroxylase was studied in rat liver microsomes. Cypro-analogues consisted of a mixture of four diasteroisomers, while MUDCA was the racemic mixture of two enantiomers. Each steroid was added to liver microsomes at concentrations ranging from 10 to 200 microM. With the exception of UCA and CA, all the bile acids inhibited cholesterol 7 alpha-hydroxylase activity. The inhibition shown by cypro-CDCA and cypro-UDCA was stronger than that observed with the corresponding natural compounds. 22S,23S cypro-UDCA exhibited an inhibitory effect which was more pronounced than that of the diasteroisomer mixture. The isomer 22R,23S was less effective and decreased cholesterol 7 alpha-hydroxylase activity in a manner comparable to that of UDCA. The effect of CDCA, UDCA and the cyclopropyl analogues was also tested with respect to HMG-CoA reductase and acylCoA cholesterol acyltransferase (ACAT) activities. ACAT was stimulated by the isomer 22S,23S cypro-UDCA but not affected by the other bile acids. No effect was observed as regards HMG-CoA reductase.