Randomised clinical trial: Lactobacillus GG modulates gut microbiome, metabolome and endotoxemia in patients with cirrhosis.

BACKGROUND: Safety of individual probiotic strains approved under Investigational New Drug (IND) policies in cirrhosis with minimal hepatic encephalopathy (MHE) is not clear. AIM: The primary aim of this phase I study was to evaluate the safety, tolerability of probiotic Lactobacillus GG (LGG) compared to placebo, while secondary ones were to explore its mechanism of action using cognitive, microbiome, metabolome and endotoxin analysis in MHE patients. METHODS: Cirrhotic patients with MHE patients were randomised 1:1 into LGG or placebo BID after being prescribed a standard diet and multi-vitamin regimen and were followed up for 8 weeks. Serum, urine and stool samples were collected at baseline and study end. Safety was assessed at Weeks 4 and 8. Endotoxin and systemic inflammation, microbiome using multi-tagged pyrosequencing, serum/urine metabolome were analysed between groups using correlation networks. RESULTS: Thirty MHE patients (14 LGG and 16 placebo) completed the study without any differences in serious adverse events. However, self-limited diarrhoea was more frequent in LGG patients. A standard diet was maintained and LGG batches were comparable throughout. Only in the LGG-randomised group, endotoxemia and TNF-α decreased, microbiome changed (reduced Enterobacteriaceae and increased Clostridiales Incertae Sedis XIV and Lachnospiraceae relative abundance) with changes in metabolite/microbiome correlations pertaining to amino acid, vitamin and secondary BA metabolism. No change in cognition was found. CONCLUSIONS: In this phase I study, Lactobacillus GG is safe and well-tolerated in cirrhosis and is associated with a reduction in endotoxemia and dysbiosis.

The effects of inflammatory cytokines on steroidogenic acute regulatory protein expression in macrophages.

OBJECTIVE: To investigate the expression of steroidogenic acute regulatory protein (StAR) in macrophages and the effects of inflammatory cytokines on StAR expression. METHODS: The macrophages isolated from ApoE knockout mice and C57BL/6J mice and RAW264.7 cells (a cell line from mouse macrophage. ATCC Number: TIB-71) were cultured in DMEM containing 10% fetal bovine serum. RAW264.7 cells were treated with different inflammatory cytokines (TNF-alpha, IFN-gamma and TGF-beta1) and 8-Br-cAMP, a cAMP analog. RT-PCR and Western blot analysis were applied to evaluate the effects of inflammatory cytokines on StAR expression. RESULTS: RT-PCR and Western blot analysis demonstrated the expression of StAR in the macrophages isolated from ApoE knockout mice, C57BL/6J mice and RAW264.7 cells. Proinflammatory cytokines TNF-alpha and IFN-gamma significantly decreased StAR mRNA and protein levels in RAW264.7 cells. The inhibition was dose- and time-dependent. In contrast, anti-inflammatory cytokine TGF-beta1 increased StAR mRNA and protein levels. At 1:15 molecular ratio, TGF-beta1 blocked the down-regulation of StAR expression mediated by TNF-alpha. cAMP also induced StAR expression in RAW264.7 cells. When the cells were co-treated with 8-Br-cAMP and TNF-alpha, 8-Br-cAMP failed to induce StAR expression. CONCLUSION: Our results provide interesting evidence that inflammatory cytokines regulate StAR expression in macrophages.

Daclizumab, a humanised monoclonal antibody to the interleukin 2 receptor (CD25), for the treatment of moderately to severely active ulcerative colitis: a randomised, double blind, placebo controlled, dose ranging trial.

BACKGROUND: An uncontrolled pilot study demonstrated that daclizumab, a humanised monoclonal antibody to the interleukin 2 receptor (CD25), might be effective for the treatment of active ulcerative colitis. METHODS: A randomised, double blind, placebo controlled trial was conducted to evaluate the efficacy of daclizumab induction therapy in patients with active ulcerative colitis. A total of 159 patients with moderate ulcerative colitis were randomised to receive induction therapy with daclizumab 1 mg/kg intravenously at weeks 0 and 4, or 2 mg/kg intravenously at weeks 0, 2, 4, and 6, or placebo. The primary end point was induction of remission at week 8. Remission was defined as a Mayo score of 0 on both endoscopy and rectal bleeding components and a score of 0 or 1 on stool frequency and physician's global assessment components. Response was defined as a decrease from baseline in the Mayo score of at least 3 points. RESULTS: Two per cent of patients receiving daclizumab 1 mg/kg (p = 0.11 v placebo) and 7% of patients receiving 2 mg/kg (p = 0.73) were in remission at week 8, compared with 10% of those who received placebo. Response occurred at week 8 in 25% of patients receiving daclizumab 1 mg/kg (p = 0.04) and in 33% of patients receiving 2 mg/kg (p = 0.30) versus 44% of those receiving placebo. Daclizumab was well tolerated. The most frequently reported adverse events in daclizumab treated patients compared with placebo treated patients were nasopharyngitis (14.6%) and pyrexia (10.7%). CONCLUSION: Patients with moderate ulcerative colitis who are treated with daclizumab are not more likely to be in remission or response at eight weeks than patients treated with placebo.

Detection of the steroidogenic acute regulatory protein, StAR, in human liver cells.

Overexpressing StAR (a mitochondrial cholesterol transporter) increases (>5-fold) the rate of 27-hydroxylation of cholesterol and the rates of bile acid synthesis in primary rat hepatocytes; suggesting that the transport of cholesterol into mitochondria is rate-limiting for bile acid biosynthesis via the CYP27A1 initiated 'acidic' pathway. Our objective was to determine the level of StAR expression in human liver and whether changes in StAR would correlate with changes in CYP27A1 activity/bile acid synthesis rates in human liver tissues. StAR mRNA and protein were detected in primary human hepatocytes and HepG2 cells by RT-PCR/Northern analysis and by Western analysis, respectively. In immunocompetition assays, liver StAR was competed away with the addition of purified human adrenal StAR. Overexpressing CYP27A1 in both cell types led to >2-fold increases in liver StAR concentration. StAR protein levels also increased approximately 2-fold with the addition of 27-hydroxycholesterol to HepG2 cell culture medium. Overexpressing StAR increased the rates of 27-hydroxylation of cholesterol/bile acid synthesis in both cell lines and increased intracellular levels of 27-hydroxycholesterol. In conclusion, human liver cells contain regulable StAR protein whose level of expression appears capable of regulating cellular cholesterol homeostasis, representing a potential therapeutic target in the management of hyperlipidemia.

Mitochondrial cholesterol transport: a possible target in the management of hyperlipidemia.

Sterol 27-hydroxylase (CYP27A1) may defend cells against accumulation of excess cholesterol, making this enzyme a possible target in the management of hyperlipidemia. The study objective was to analyze cholesterol homeostatic responses to increases in CYP27A1 activity in HepG2 cells and primary human hepatocytes. Increasing CYP27A1 activity by increasing enzyme expression led to significant increases in bile acid synthesis with compensatory increases in HMG-CoA reductase (HMGR) activity/protein, LDL receptor (LDLR) mRNA, and LDLR-mediated cholesterol uptake. Under these conditions, only a small increase in cellular 27-hydroxycholesterol (27OH-Chol) concentration was observed. No changes were detected in mature sterol regulatory element-binding proteins (SREBP) 1 or 2. Increasing CYP27A1 activity by increasing mitochondrial cholesterol transport (i.e., substrate availability) led to greater increases in bile acid synthesis with significant increases in cellular 27OH-Chol concentration. Mature SREBP 2 protein decreased significantly with compensatory decreases in HMGR protein. No change was detected in mature SREBP 1 protein. Despite increasing 27OH-Chol and lowering SREBP 2 protein concentrations, LDLR mRNA increased significantly, suggesting alternative mechanisms of LDLR transcriptional regulation. These findings suggest that regulation of liver mitochondrial cholesterol transport represents a potential therapeutic strategy in the treatment of hyperlipidemia and atherosclerosis.

Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis.

The initial and rate-limiting step in the classic pathway of bile acid biosynthesis is 7alpha-hydroxylation of cholesterol, a reaction catalyzed by cholesterol 7alpha-hydroxylase (CYP7A1). The effect of CYP7A1 overexpression on cholesterol homeostasis in human liver cells has not been examined. The specific aim of this study was to determine the effects of overexpression of CYP7A1 on key regulatory steps involved in hepatocellular cholesterol homeostasis, using primary human hepatocytes (PHH) and HepG2 cells. Overexpression of CYP7A1 in HepG2 cells and PHH was accomplished by using a recombinant adenovirus encoding a CYP7A1 cDNA (AdCMV-CYP7A1). CYP7A1 overexpression resulted in a marked activation of the classic pathway of bile acid biosynthesis in both PHH and HepG2 cells. In response, there was decreased HMG-CoA-reductase (HMGR) activity, decreased acyl CoA:cholesterol acyltransferase (ACAT) activity, increased cholesteryl ester hydrolase (CEH) activity, and increased low-density lipoprotein receptor (LDLR) mRNA expression. Changes observed in HMGR, ACAT, and CEH mRNA levels paralleled changes in enzyme specific activities. More specifically, LDLR expression, ACAT activity, and CEH activity appeared responsive to an increase in cholesterol degradation after increased CYP7A1 expression. Conversely, accumulation of the oxysterol 7alpha-hydroxycholesterol in the microsomes after CYP7A1 overexpression was correlated with a decrease in HMGR activity.

Expression of sterol 12alpha-hydroxylase alters bile acid pool composition in primary rat hepatocytes and in vivo.

BACKGROUND & AIMS: The rate of 12alpha-hydroxylation of bile acid intermediates is believed to determine the ratio of cholic acid (CA) to chenodeoxycholic acid (CDCA) biosynthesis and the overall hydrophobicity of the bile acid pool. The aim of this study was to determine the effects of the level of expression of sterol 12alpha-hydroxylase (CYP8b1) and cholesterol 7alpha-hydroxylase (CYP7a1) on rates of CA biosynthesis and bile acid pool composition. METHODS: Expression of CYP8b1 and CYP7a1 was accomplished through infection of primary rat hepatocytes (PRH) or intact male SD rats with replication-defective recombinant adenoviruses encoding either CYP8b1 or CYP7a1. RESULTS: Increased expression of CYP7a1 over basal levels in PRH dramatically increased bile acid biosynthesis (586% +/- 82%, P < 0.001) but did not alter the ratio of CA to CDCA. Conversely, increased expression of CYP8b1 in vitro had no significant effect on the rates of total bile acid synthesis but significantly increased (4.1-fold) the rates of CA biosynthesis, resulting in an increase in the CA-CDCA ratio from 1:6.6 to 2.8:1. In whole rats, increased CYP8b1 expression over basal levels markedly increased the CA in the bile acid pool from 36% +/- 3.4% to 50% +/- 2.9% in 5 days. CDCA and its muricholic acid derivatives decreased from 64% +/- 3.4% to 50% +/- 2.9%. CONCLUSIONS: Increased expression of CYP8b1 led to a marked increase in CA biosynthesis both in PRH and in whole animals. CYP8b1 is capable of 12alpha-hydroxylating bile acid intermediates from both the classic and acidic pathways.

Regulation of multidrug resistance 2 P-glycoprotein expression by bile salts in rats and in primary cultures of rat hepatocytes.

Biliary phospholipid secretion is tightly coupled to the secretion of free cholesterol and bile salts. The secretion of phospholipids across the canalicular membrane of hepatocytes occurs via the multidrug resistance 2 (mdr2) P-glycoprotein (Pgp). The mechanism underlying the coupling of bile salt and phospholipid secretion has not been elucidated. The aims of this study were to determine the effects of bile acid structure on the expression of mdr2 in vitro and in vivo. Under optimal culture conditions, taurine-conjugated bile acids (50 micromol/L) increased mdr2 messenger RNA (mRNA) levels in the following order: taurocholate (TCA) (288 +/- 36%, P <. 005) = taurodeoxycholate (TDCA) (276 +/- 36%, P <.025) > taurochenodeoxycholate (TCDCA) (216 +/- 34%, P <.025) > tauroursodeoxycholate (TUDCA) (175 +/- 28%, P <.05) of control levels. The increase in mdr2 mRNA levels by TCA was both time and concentration dependent. Cholate feeding to rats with intact enterohepatic circulation increased mdr2 transcriptional activity by 4-fold and protein mass by 1.9-fold. Chronic biliary diversion (CBD) decreased mdr2 mRNA levels to 66 +/- 9% (P <.025) of sham-operated controls. Intraduodenal infusion of TCA for 48 hours in CBD rats caused a significant increase in mdr2 mRNA levels (224%) as compared with CBD controls. A diet high in cholesterol (4%) decreased mdr2 mRNA levels to 57% +/- 2 (P <.001) of pair-fed controls. Squalestatin (1 micromol/L), an inhibitor of cholesterol biosynthesis, increased mdr2 mRNA levels by 8.8-fold (P <.005) in hepatocyte cultures after 24 hours. In conclusion, in the rat, bile acids up-regulated mdr2 transcriptional activity whereas cholesterol decreased mdr2 mRNA both in vitro and in vivo.

Regulation of sterol 12alpha-hydroxylase and cholic acid biosynthesis in the rat.

BACKGROUND & AIMS: Sterol 12alpha-hydroxylase (CYP8b1) is required for the biosynthesis of cholic acid (CA) and hence helps determine the ratio of CA to chenodeoxycholic acid (CDCA) in bile. This study examined the in vivo regulation of CYP8b1 in the rat by bile acids, cholesterol, and thyroxine. METHODS: The specific activities (SAs), messenger RNA (mRNA) levels, and transcriptional activities of CYP8b1 were determined in intact rats and rats with biliary diversion. RESULTS: CA, CDCA, and deoxycholic acid (DCA), fed as a supplement to the diet, down-regulated CYP8b1 SAs by 99% +/- 0%, 72% +/- 10%, and 98% +/- 1%, respectively. Under these same conditions, mRNA levels decreased by 93% +/- 7%, 60% +/- 11%, and 93% +/- 4%, respectively. Intraduodenal infusion of taurocholate (36 micromol/h. 100 g rat(-1)) decreased SAs and mRNA levels by 63% +/- 8% and 74% +/- 8%, respectively. Ursodeoxycholic acid (UDC) and hyocholic acid (HC) feeding increased CYP8b1 SAs by 119% +/- 21% and 65% +/- 18%, respectively. CA feeding decreased CYP8b1 transcriptional activity by 72%. Complete biliary diversion increased CYP8b1 SAs and mRNA levels by 150% +/- 30% and 287% +/- 51%, respectively. Cholesterol feeding decreased CYP8b1 mRNA by 39% +/- 8%. In intact rats, a single injection of thyroid hormone eliminated CYP8b1 activity. CONCLUSIONS: CYP8b1 is transcriptionally down-regulated by hydrophobic but not hydrophilic bile acids. Cholesterol feeding and a single thyroid hormone injection repressed CYP8b1 in the face of induction of cholesterol 7alpha-hydroxylase (CYP7a1 by the new nomenclature) SAs. These results suggest that cholesterol, thyroid hormone, and hydrophobic bile acids are important regulators of CYP8b1 and consequently of the bile acid pool composition.

Regulation of bile acid biosynthesis.

This article provides a review of the pathways through which cholesterol is degraded to bile acids. Regulation of key enzymes in the bile acid biosynthestic pathways is discussed. The important role of these pathways in the maintenance of cholesterol homeostasis and the possible therapeutic implications for the treatment of hypercholesterolemia are emphasized.

Regulation of hepatic neutral cholesteryl ester hydrolase by hormones and changes in cholesterol flux.

To understand molecular events in regulation of hepatic neutral cholesteryl ester hydrolase (EC3.1.1.13; CEH), catalytic activity, protein mass, and mRNA levels were measured in rats with various perturbations of hepatic cholesterol metabolism. Cholesterol feeding decreased activity (56 +/- 2%), mass (44 +/- 2%), and mRNA (14 +/- 3%). The cholesterol precursor mevalonate also decreased activity (42 +/- 6%), mass (76 +/- 3%), and mRNA (23 +/- 16%). Inhibition of cholesterol biosynthesis by lovastatin increased activity (65 +/- 12%) and mRNA (31 +/- 24%). Stimulation of cholesterol efflux by chronic biliary diversion increased activity (138 +/- 34%), mass (29 +/- 7%), and mRNA (146 +/- 28%). Chenodeoxycholate feeding decreased activity (46 +/- 6%) and mRNA (26 +/- 12%). These data suggest rational regulation of CEH in response to changes in cholesterol flux through the liver. In primary hepatocytes, steady-state mRNA markedly decreased during 72-h cultures and addition of L-thyroxine and dexamethasone synergistically maintained mRNA levels near control values. Lovastatin increased mRNA levels by 103 +/- 15%. Taurocholate and phorbol 12-myristate 13-acetate suppressed mRNA (61 +/- 4% and 49 +/- 13%, respectively), suggesting that protein kinase C mediated effects of bile acids on CEH mRNA levels. These data suggest regulation of CEH by hormones and signal transduction in addition to changes in cholesterol flux.

Effect of bile acid composition and manipulation of enterohepatic circulation on leptin gene regulation.

In the rat, the ob gene product, leptin, putatively regulates energy balance via appetite control and energy expenditure. Bile acids in the intestinal lumen are necessary for efficient absorption of dietary lipids and may trigger the release of regulatory peptides. To investigate whether bile acids play a role in leptin gene expression, we altered the bile acid pool and then measured leptin mRNA levels in adipose tissue. Rats fed cholic acid (1% of chow wt/wt) for 2 weeks did not gain weight as rapidly as pair-fed control animals. Despite the lower weight, normalized leptin mRNA levels were 24% greater in cholic acid-fed rats compared with controls. Conversely, cholestyramine, a bile acid sequestrant, in chow (5% wt/wt) resulted in a 26% decline in leptin mRNA. Ligation of the common bile duct or chronic biliary diversion, experimental manipulations that decreased the intestinal concentration of bile salts, decreased leptin gene expression by 30% and 50%, respectively. A fluid and electrolyte (F/E) solution with and without taurocholate (36 micromol/h x 100 g rat[-1]) was then infused for 12 hours into the duodenum in animals with chronic biliary diversion. Taurocholate infusion resulted in a fourfold increase in steady-state adipocyte leptin mRNA levels compared with F/E infusion. Intravenous infusion of taurocholate or incubation of cultured adipocytes with taurocholate had no effect on leptin mRNA levels. We conclude that bile acids upregulate leptin gene expression indirectly, probably via effects on the absorption of dietary lipids or the release of neurohumoral mediators.

Quantitative estimations of the contribution of different bile acid pathways to total bile acid synthesis in the rat.

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.

Induction of sodium-dependent bile acid transporter messenger RNA, protein, and activity in rat ileum by cholic acid.

BACKGROUND & AIMS: The ileal sodium-dependent bile acid transporter reclaims bile acids from the intestinal lumen to preserve their enterohepatic recirculation. The present studies sought to determine the possible role of enteric bile acids in the molecular regulation of the apical bile acid transporter in rat ileal mucosa. METHODS: Paired rats were fed a control diet or control diet plus cholic acid (1%) or ursodeoxycholic acid (1%) for 10 days. Other paired rats underwent biliary diversion for 72 hours, followed by intraduodenal infusion of taurocholate or fluid/electrolytes. Transporter protein, messenger RNA (mRNA), and activity were determined in the distal 15 cm of ileal mucosa. RESULTS: Transporter protein and mRNA levels in cholic acid-fed rats increased approximately threefold above levels in paired rats fed the control diet (P < 0.02). Similarly, sodium-dependent [3H]taurocholate uptake into membrane vesicles from cholic acid-fed rats increased twofold above uptake into vesicles from control-fed rats because of a twofold increase in maximal transport velocity. In biliary-diverted rats (72-96 hours), transporter protein decreased to 57% +/- 5% of paired controls with intact enterohepatic circulation (P < 0.0001). The intraduodenal infusion of taurocholate (24 hours) in biliary-diverted rats resulted in a time-dependent reinduction of transporter protein expression (3.5-fold). CONCLUSIONS: The expression of the ileal apical bile acid transporter is induced at a pretranslational level by free or taurine-conjugated cholic acid within the small intestine.

Effect of enteral versus parenteral nutrition on leptin gene expression and release into the circulation.

Leptin, a putative satiety hormone in rodents, is acutely regulated by fasting and refeeding. To determine the role of satiety hormones that are secreted by the gastrointestinal tract on leptin regulation, leptin mRNA and serum concentrations were measured after feeding rats similar calories with standard chow or infusion of total parenteral nutrition into the duodenum or intravenously. We have demonstrated that leptin gene expression and hormone secretion into the circulation are stimulated equally in the three experimental paradigms; it is unlikely that satiety factors secreted by the intestinal tract play a significant role in leptin regulation. Furthermore, intravenous infusion of individual components of TPN demonstrated that intravenous glucose infusion was mostly responsible for stimulation of the leptin gene and hormone secretion.

Hormonal regulation of cholesterol 7alpha-hydroxylase specific activity, mRNA levels, and transcriptional activity in vivo in the rat.

In primary cultures of rat hepatocytes, transcription of the cholesterol 7alpha-hydroxylase gene is induced synergistically by glucocorticoid and thyroid hormones. The objective of the present study was to evaluate the role of endogenous glucocorticoid and thyroid hormones in the maintenance of cholesterol 7alpha-hydroxylase gene expression in vivo. Male Sprague-Dawley rats underwent adrenalectomy (A), thyroidectomy (T), adrenalectomy + thyroidectomy (A + T), hypophysectomy (H), or sham surgery (paired controls). Ten days post surgery, livers were harvested and choles terol 7alpha-hydroxylase specific activity, steady-state mRNA levels, and transcriptional activity were determined. Serum corticosterone levels were <2% of paired controls in A, A + T, and H rats. Free thyroxine index was <32% of paired controls in rats with T and H. When compared to sham-operated controls, A + T and H led to decreases in cholesterol 7alpha-hydroxylase specific activities of 44 +/- 8% and 57 +/- 3%, respectively (P < 0.03 and < 0.05). Similar changes were observed in cholesterol 7alpha-hydroxylase steady-state mRNA levels, which decreased by 43 +/- 10% (P < 0.001) and 56 +/- 19% (P < 0.05), respectively. Cholesterol 7alpha-hydroxylase transcriptional activity in A + T and H rats decreased by 34 +/- 11% (P < 0.01) and 61 +/- 4% (P < 0.001), respectively. The observed decreases were greater after H than after A + T, suggesting the possibility that another pituitary hormone plays a role in regulation of cholesterol 7alpha-hydroxylase. Thyroidectomy alone led to a decrease in cholesterol 7alpha-hydroxylase specific activity of 37 +/- 7% (P < 0.05) and a trend toward decreased steady-state mRNA levels (21 +/- 12%; P = ns). Adrenalectomy did not significantly decrease cholesterol 7alpha-hydroxylase specific activity or mRNA levels. Neither thyroidectomy nor adrenalectomy alone affected transcriptional activity. We conclude that under physiologic circumstances, full expression of the cholesterol 7alpha-hydroxylase gene requires synergistic action of glucocorticoids and thyroid hormone.

Transcriptional regulation of hepatic sterol 27-hydroxylase by bile acids.

The study objective was to determine whether and to what extent sterol 27-hydroxylase, the initial step in the "acidic" pathway of bile acid biosynthesis, is regulated by bile acids. Rats were fed diets supplemented with cholestyramine (CT, 5%), cholate (CA, 1%), chenodeoxycholate (CDCA, 1%), or deoxycholate (DCA, 0.25%). When compared with paired controls, sterol 27-hydroxylase and cholesterol 7 alpha-hydroxylase specific activities increased after CT administration by 188 +/- 20% (P < 0.05) and 415 +/- 36% (P < 0.01), respectively. Similarly, mRNA levels increased by 159 +/- 14% (P < 0.05) and 311 +/- 106% (P < 0.05), respectively. Feeding CA, CDCA, or DCA decreased sterol 27-hydroxylase specific activity to 57 +/- 6, 61 +/- 8, and 74 +/- 8% of controls, respectively (P < 0.05). By comparison, the specific activity of cholesterol 7 alpha-hydroxylase decreased to 46 +/- 7 , 32 +/- 10, and 26 +/- 8% (P = 0.001). mRNA levels and transcriptional activities for sterol 27-hydroxylase and cholesterol 7 alpha-hydroxylase transcriptional activity were changed to the same extent as the specific activities after CT or bile acid feeding. We conclude that sterol 27-hydroxylase and cholesterol 7 alpha-hydroxylase are subject to negative feedback regulation by hydrophobic bile acids at the level of transcription. However, the responses of sterol 27-hydroxylase to manipulation of the bile acid pool are less prominent than those of cholesterol 7 alpha-hydroxylase. During the diurnal cycle the specific activities of sterol 27-hydroxylase and cholesterol 7 alpha-hydroxylase changed in tandem, suggesting that both may be under control of glucocorticoids.

Hep G2 cells: a model for studies on regulation of human cholesterol 7alpha-hydroxylase at the molecular level.

The present study examines the feedback control governing human cholesterol 7alpha-hydroxylase mRNA expression in the human hepatoblastoma cell line, Hep G2. Glycochenodeoxycholate (GCDC) and glycodeoxycholate, hydrophobic bile salts, decreased cholesterol 7alpha-hydroxylase mRNA levels and bile acid synthesis in a concentration-dependent (76 +/- 8%, P<0.001, and 48 +/- 3%, P<0.01, respectively) and time-dependent manner. Cholesterol 7alpha-hydroxylase mRNA levels were repressed with a half-maximal inhibitory concentration of <12.5 microM by GCDC and a half-life of 30 min by 100 microM of the bile acid. The addition of actinomycin D (10 microgram/ml) alone or in combination with GCDC (100 microM) led to similar concentration-and time-dependent suppression of cholesterol 7alpha-hydroxylase mRNA. Glycocholate (100 microM), not internalized based on lack of uptake of a fluorescent cholate analogue, had no effect on cholesterol 7alpha-hydroxylase mRNA or total bile acid synthesis. In cultures transfected with a rat cholesterol 7alpha-hydroxylase promoter construct, reporter gene activity was decreased (31%, P<0.01) by GCDC (100 microM). Hep G2 cells maintain the intracellular machinery to express and rapidly regulate human cholesterol 7alpha-hydroxylase by hydrophobic bile acids. These data suggest that Hep G2 cells will support functional studies of the human cholesterol 7alpha-hydroxylase gene.

Failure of intravenous infusion of taurocholate to down-regulate cholesterol 7 alpha-hydroxylase in rats with biliary fistulas.

BACKGROUND/AIMS: The decrease in cholesterol 7 alpha-hydroxylase induced by intraduodenal infusion of taurocholate in bile fistula rats may be indirect, i.e., mediated through release or absorption of an intestinal factor in response to the presence of bile salts in the intestine. The aim of this study was to determine if negative feedback regulation of cholesterol 7 alpha-hydroxylase can be shown when equimolar concentrations of taurocholate are administered intravenously, thus bypassing the intestine. METHODS: After 96 hours of biliary diversion, taurocholate (36 mumol.h-1.100 g, rat-1) was infused into the rats either intravenously or intraduodenally for the final 24 hours. Livers were then harvested for analysis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase specific activity, cholesterol 7 alpha-hydroxylase specific activity, messenger RNA levels, and transcriptional activity. RESULTS: Intraduodenally administered taurocholate significantly decreased HMG-CoA reductase and cholesterol 7 alpha-hydroxylase specific activity by more than 50% and cholesterol 7 alpha-hydroxylase steady-state messenger RNA levels and transcriptional activity by 50%-75%. In contrast, intravenous administration of taurocholate failed to down-regulate either cholesterol 7 alpha-hydroxylase or HMG-CoA reductase. CONCLUSIONS: Passage of taurocholate through the intestine strongly potentiates negative feedback regulation of cholesterol 7 alpha-hydroxylase. A putative intestinal factor, released or absorbed in the presence of bile acids in the intestinal lumen, may play a role in the regulation of bile acid synthesis.

Post-transcriptional regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase in rats with subtotal nephrectomy.

Chronic renal failure is associated with hyperlipidemia and atherosclerosis. The mechanism responsible for the observed increase of serum cholesterol in chronic renal disease is not certain. The objective of the present study was to characterize the effect of induced renal failure on 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) and cholesterol 7 alpha-hydroxylase, the two rate determining enzymes of the cholesterol and bile acid biosynthetic pathways, respectively. Studies were carried out in rats with subtotal (75%) nephrectomy, which resulted in a marked elevation of blood urea nitrogen (371 +/- 44% of control, P < 0.001), and was accompanied by significant increases in the levels of serum cholesterol (133 +/- 7%, P < 0.005) and triglycerides (185 +/- 25, P < 0.01). In nephrectomized rats, an increase in the specific activity of HMG-CoA reductase (219 +/- 30% above control levels, P < 0.02) was observed. This increase occurred in the presence of elevated hepatic microsomal cholesterol concentrations (150 +/- 13% of controls, P < 0.01). Surprisingly, the increase in HMG-CoA reductase specific activity was not associated with parallel increases in HMG-CoA reductase steady-state mRNA levels and gene transcriptional activity. These uremic rats also exhibited a marked increase in the specific activity of cholesterol 7 alpha-hydroxylase (240 +/- 559% of controls, P < 0.05). There was no concomitant increase in cholesterol 7 alpha-hydroxylase steady-state mRNA levels or gene transcriptional activity. The factors responsible for the observed increases in HMG-CoA reductase and cholesterol 7 alpha-hydroxylase specific activity in renal failure remain to be determined.(ABSTRACT TRUNCATED AT 250 WORDS)