Glutamine attenuates obstructive cholestasis in rats via farnesoid X receptor-mediated regulation of Bsep and Mrp2.

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.

Diet1 is a regulator of fibroblast growth factor 15/19-dependent bile acid synthesis.

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.

The COUP-TFII variant lacking a DNA-binding domain inhibits the activation of the Cyp7a1 promoter through physical interaction with COUP-TFII.

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.

Fibroblast growth factor 7 inhibits cholesterol 7α-hydroxylase gene expression in hepatocytes.

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.

Sesquiterpenoids from Atractylodes macrocephala act as farnesoid X receptor and progesterone receptor modulators.

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.

Forkhead box transcription factor O1 inhibits cholesterol 7alpha-hydroxylase in human hepatocytes and in high fat diet-fed mice.

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.

Oxidized low-density lipoprotein increases interleukin-8 production in human gingival epithelial cell line Ca9-22.

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.

Transthyretin Regulated by linc00657/miR-205-5p Promoted Cholesterol Metabolism by Inducing SREBP2-HMGCR and Inhibiting LXRα-CYP7A1.

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.

Hepatic cholesterol accumulation ascribed to the activation of ileum Fxr-Fgf15 pathway inhibiting hepatic Cyp7a1 in high-fat diet-induced obesity rats.

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.

Effects of dietary crude oil exposure on molecular and physiological parameters related to lipid homeostasis in polar cod (Boreogadus saida).

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-ß], palmitoyl-CoA oxidase [aox1], cytochrome P4507A1 [cyp7α1]), reproduction (vitellogenin [vtg-ß], 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.

Ketoconazole blocks bile acid synthesis in hepatocyte monolayer cultures and in vivo in rat by inhibiting cholesterol 7 alpha-hydroxylase.

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.

Activation of the silent endogenous cholesterol-7-alpha-hydroxylase gene in rat hepatoma cells: a new complementation group having resistance to 25-hydroxycholesterol.

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.

Activation of caspase-8 and caspase-12 pathways by 7-ketocholesterol in human retinal pigment epithelial cells.

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.

The effects of 6-azacholest-4-en-3 beta-ol-7-one, an inhibitor of cholesterol 7 alpha-hydroxylase, on cholesterol metabolism and bile acid synthesis in primary cultures of rat hepatocytes.

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.

Effect of prostaglandins E1 and F1alpha on the activities of cholesteryl ester synthetase and cholesteryl ester hydrolases of pigeon aorta in vitro.

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.

Effect of natural and structurally modified bile acids on cholesterol metabolizing enzymes in rat liver microsomes. II.

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.

Effect of natural and structurally modified bile acids on cholesterol metabolizing enzymes in rat liver microsomes.

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.

Effects of superoxide dismutase on cholesterol 7 alpha-hydroxylation in swine.

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 suppressive effect of the catatoxic steroid, pregnenolone-16alpha-carbonitrile, on liver microsomal cholesterol-7alpha-hydroxlyase.

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.

Inhibition of cholesterol and fatty acid biosynthesis in liver enzymes and chicken hepatocytes by polar fractions of garlic.

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.