Search

The initial and rate-limiting step in the classic pathway of bile acid biosynthesis is 7α-hydroxylation of cholesterol, a reaction catalyzed by cholesterol 7α-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 7α-hydroxycholesterol in the microsomes after CYP7A1 overexpression was correlated with a decrease in HMGR activity.

In the liver, sterol 27-hydroxylase (CYP27) participates in the classic and alternative pathways of bile acid biosynthesis from cholesterol (Chol). In extrahepatic tissues, CYP27 converts intracellular Chol to 27-hydroxycholesterol (27OH-Chol), which may regulate the activity of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA-R). This study attempts to better define the role of CYP27 in the maintenance of Chol homeostasis in hepatic and extrahepatic cells by overexpressing CYP27 in Hep G2 cells and Chinese hamster ovary (CHO) cells through infection with a replication-defective recombinant adenovirus encoding for CMV-CYP27. After infection, CYP27 mRNA and protein levels increased dramatically. CYP27 specific activity also increased two- to fourfold in infected cells ( P ≤ 0.02), with a marked increase in conversion of [14C]Chol to [14C]27OH-Chol (∼150%; P ≤ 0.01). Accumulation of 27OH-Chol in CHO cells was associated with a 50% decrease in HMG-CoA-R specific activity ( P ≤ 0.02). In infected Hep G2 cells, the significant increase in bile acid synthesis (46%; P ≤ 0.006), which prevented the accumulation of intracellular 27OH-Chol, resulted in increased HMG-CoA-R activity (183%; P ≤ 0.02). Overexpression of CYP27 in Hep G2 cells also increased acyl CoA-cholesterol acyltransferase (71%, P ≤ 0.02) and decreased cholesteryl ester hydrolase (55%, P ≤ 0.02). In conclusion, CYP27 generates different physiological responses depending on cell type and presence or absence of bile acid biosynthetic pathways.

In vitro and in vivo studies have shown that the sterol 27-hydroxylase (CYP27) gene is transcriptionally repressed by hydrophobic bile acids. The molecular mechanism(s) of repression of CYP27 by bile acids is unknown. To identify the bile acid responsive element (BARE) and transcription factor(s) that mediate the repression of CYP27 by bile acids, constructs of the CYP27 5'-flanking DNA were linked to either the CAT or luciferase reporter gene and transiently transfected into primary rat hepatocytes. Taurocholate (TCA), taurodeoxycholate (TDCA) and taurochenodeoxycholate (TCDCA) significantly reduced CAT activities of the -840/+23, -329/+23, and -195/+23 mCAT constructs. A -76/+23 construct showed no regulation by bile acids. When a DNA fragment (-110/-86) from this region was cloned in front of an SV 40 promoter it showed down-regulation by TDCA. 'Super'-electrophoretic mobility shift assays (EMSA) indicated that both HNF1alpha and C/EBP bind to the -110 to -86 bp DNA fragment. Recombinant rat HNF1alpha and C/EBPalpha competitively bound to this DNA fragment. 'Super'-EMSA showed that TDCA addition to hepatocytes in culture decreased HNF1alpha, but not C/EBP, binding to the -110/-86 bp DNA fragment. A four base pair substitution mutation (-103 to -99) in this sequence eliminated TCA and TDCA regulation of the (-840/+23) construct. The substitution mutation also eliminated (>95%) HNF1alpha, but not C/EBP, binding to this DNA fragment. We conclude that bile acids repress CYP27 transcription through a putative BARE located between -110 and -86 bp of the CYP27 promoter. The data suggest that bile acids repress CYP27 transcriptional activity by decreasing HNF1alpha binding to the CYP27 promoter.

We have recently shown that taurocholate (TCA) represses the transcriptional activity of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme of the bile acid biosynthetic pathway, through a protein kinase C (PKC)-dependent mechanism in primary cultures of rat hepatocytes. The present studies sought to determine the mechanisms by which bile acids activate hepatic PKC activity and the consequences of this activation on isoform distribution and cholesterol 7 alpha-hydroxylase mRNA levels. TCA (12.5-100 microM for 15 min) increased membrane-associated "classic" isoenzyme cPKC-alpha and "novel" isoenzymes nPKC-delta, and nPKC by two- to sixfold. Membrane-associated PKC progressively increased, and cytosolic PKC decreased, for 1 h after the addition of TCA (50 microM); after 24 h whole cell cPKC-alpha, nPKC-delta, and nPKC were downregulated by 35-55% compared with untreated controls. In a reconstituted assay system, TCA or taurodeoxycholate (10-100 microM) increased calcium-dependent and -independent PKC activity by three- and fourfold, respectively. Taurine-conjugated bile acids stimulated PKC activity in proportion to their hydrophobicity index (r = 0.99). Finally, cholesterol 7 alpha-hydroxylase mRNA was repressed > 75% by phorbol 12-myristate 13-acetate (100 nM for 3 h), a nonselective activator of PKC isoforms. In contrast, selective cPKC-alpha activation with thymeleatoxin (100 nM for 3 h) had no significant effect on cholesterol 7 alpha-hydroxylase mRNA levels. We conclude that bile acids activate hepatocellular PKC, resulting in sequential redistribution and down-regulation of calcium-dependent and -independent isoforms. The calcium-independent PKC isoforms may mediate the repression of cholesterol 7 alpha-hydroxylase mRNA by TCA.

The importance of cholesterol and “oxysterols” in the regulation of cholesterol 7 alpha-hydroxylase is not clear. Previous in vivo studies suggest that cholesterol may up-regulate cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, but these studies are open to question as they were carried out in whole animals. Therefore, we used primary rat hepatocytes, cultured in serum-free medium, to determine the effects of cholesterol on the regulation of cholesterol 7 alpha-hydroxylase. Squalestatin, a specific squalene synthase inhibitor, was used to block sterol but not isoprenoid biosynthesis in this system. Squalestatin (1 microM) decreased cholesterol 7 alpha-hydroxylase specific activity to undetectable levels and decreased steady-state mRNA and transcriptional activity to 13% and 47% of controls, respectively. Mevalonolactone (2 mM) failed to restore cholesterol 7 alpha-hydroxylase specific activity or steady-state mRNA levels in squalestatin-treated cells. Addition of cholesterol, delivered in beta-cyclodextrin, to squalestatin-treated cells restored cholesterol 7 alpha-hydroxylase specific activity and steady-state mRNA to control levels in a concentration (25 microM to 200 microM) -dependent manner. In contrast, the individual addition of selected “oxysterols” (5-cholesten-3 beta, 7 alpha-diol; 5 alpha-cholestan-3 beta, 6 alpha-diol; cholestan-3 beta, 5 alpha,6 beta-triol; 5-(25R)-cholesten-3 beta,26-diol, all at 50 microM) failed to restore cholesterol 7 alpha-hydroxylase mRNA levels in squalestatin-treated cells. These experiments provide evidence that cholesterol rather than “oxysterols” regulate cholesterol 7 alpha-hydroxylase gene expression. Squalestatin (1 microM) treatment increased HMG-CoA reductase specific activity by 229% of controls. Addition of cholesterol (200 microM), but not mevalonolactone (2 mM), to squalestatin-treated cells decreased HMG-CoA reductase specific activity to 19% of control. The primary rat hepatocyte culture system in conjunction with a specific squalene synthetase inhibitor should be a useful model for elucidating the mechanism of regulation of cholesterol 7 alpha-hydroxylase gene expression by sterols.

The role of bile acids in the regulation of cholesterol 7 alpha-monooxygenase (EC 1.14.13.17) was characterized using primary cultures of rat hepatocytes supplemented with dexamethasone and thyroxine. Taurocholate and taurodeoxycholate (50 microM) repressed cholesterol 7 alpha-hydroxylase mRNA to 44 +/- 9 and 52 +/- 4%, respectively, of control values. Repression by these natural, relatively hydrophobic bile acids was concentration dependent, with an IC50 of about 50 microM, and time dependent with a t1/2 for repression of 22 h. In contrast, two natural hydrophilic bile acids, tauroursodeoxycholate and taurohyodeoxycholate, had no effect on cholesterol 7 alpha-hydroxylase mRNA levels. Taurochenodeoxycholate and taurolithocholate also had no effect, but these hydrophobic bile acids were rapidly hydroxylated to more hydrophilic bile acids. Hydrophilic bile acid analogues (nor (C23) bile acids and beta-hydroxy epimers) repressed cholesterol 7 alpha-hydroxylase mRNA less potently than their corresponding and more hydrophobic C24 or alpha-hydroxy derivatives. Cholesterol 7 alpha-hydroxylase specific activity was decreased by taurocholate or taurodeoxycholate (50 microM) to 26 +/- 9 and 56 +/- 3% of control, respectively; its transcriptional activity was repressed to 52 +/- 5% of control by taurocholate (50 microM). The addition of cholesterol or the induction of cholesterol biosynthesis did not influence repression of cholesterol 7 alpha-hydroxylase mRNA levels by taurocholate. Based on several lines of evidence, cAMP was not involved in bile acid-induced repression. In rat hepatocytes cultured under conditions in which cholesterol 7 alpha-hydroxylase gene expression is maintained at in vivo levels, hydrophobic bile acids repress this enzyme at the level of gene transcription independently of cholesterol availability.

Cholesterol 7 alpha-hydroxylase, the rate-determining enzyme in the bile acid biosynthesis pathway, is regulated in a negative feedback manner by hydrophobic bile salts returning to the liver via the portal circulation. The role of cholesterol in the regulation of cholesterol 7 alpha-hydroxylase and the interrelationship between the cholesterol and bile acid biosynthesis pathways remain controversial. The objective of the present study was to define the role of cholesterol in the regulation of cholesterol 7 alpha-hydroxylase and determine the molecular level of its control. In order to avoid intestinal or intravenous administration of cholesterol, we manipulated the flow of cholesterol within the hepatocytes by decreasing cholesterol synthesis with lovastatin in bile fistula rats (bile acid synthesis is up-regulated), or by increasing cholesterol supply by administering mevalonate, a precursor of cholesterol, to rats with intact enterohepatic circulation (bile acid synthesis is normal). In the first series of studies, lovastatin was administered as a single intravenous bolus (10 mg/kg) to rats with chronic bile fistula and to rats with intact enterohepatic circulation (cholesterol and bile acid synthesis is normal). Three hours after lovastatin administration, cholesterol 7 alpha-hydroxylase specific activity, enzyme mass, mRNA, and gene transcriptional activity were decreased by 35%, 32%, 56%, and 34%, respectively, in rats with chronic bile fistula. In rats with intact enterohepatic circulation, lovastatin administration resulted in a similar decrease (34%) of cholesterol 7 alpha-hydroxylase specific activity.(ABSTRACT TRUNCATED AT 250 WORDS)

In primary cultures of adult rat hepatocytes the level of cholesterol 7 alpha-hydroxylase steady-state mRNA markedly decreased by 72 h. However, the addition of L-thyroxine (T4) and dexamethasone synergistically returned cholesterol 7 alpha-hydroxylase steady-state mRNA levels near to that of cholestyramine-fed animals. The maximal responses to T4 and dexamethasone in serum-free medium were at 1.0 and 0.1 microM, respectively. The addition of T4 in combination with dexamethasone resulted in an 11-fold increase in transcriptional activity of the cholesterol 7 alpha-hydroxylase gene as compared to no addition controls. The specific activities of cholesterol 7 alpha-hydroxylase in microsomes prepared from cultures treated with dexamethasone and T4 were 1.56 +/- 1.17 nmol/h/mg protein which is similar to that of intact liver (1.70 +/- 0.062 nmol/h/mg protein), but lower than cholestyramine-fed animals. Cholesterol 7 alpha-hydroxylase activity was not detectable (less than 0.020 nmol/h/mg protein) at 72 h in cultures without the addition of both dexamethasone and T4. In the presence of optimal concentrations of dexamethasone and T4, glucagon (0.2 microM), or dibutyryl cAMP (50 microM) decreased (90%) cholesterol 7 alpha-hydroxylase mRNA within 6 h. Transcriptional activity decreased (62%) in 6 h following the addition of glucagon (0.2 microM) to the culture medium. The results reported in this paper suggest an important role for multiple hormones in the regulation of cholesterol 7 alpha-hydroxylase in the liver.

Taurocholate, a relatively hydrophobic bile salt, is a potent down-regulator of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase (C7 alpha H), the rate-determining enzymes of the cholesterol and bile acid biosynthetic pathways, respectively. Inhibition of cholesterol synthesis with a bolus dose of mevinolin (lovastatin) a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, profoundly decreases the specific activity of cholesterol 7 alpha-hydroxylase and rate of bile acid synthesis in rats with complete biliary diversion. It is therefore conceivable that taurocholate may suppress cholesterol 7 alpha-hydroxylase primarily by down-regulating the activity of HMG-CoA reductase. To test this hypothesis, taurocholate was coinfused simultaneously to rats with chronic bile fistula with mevalonate (administered as mevalonolactone), an intermediate in the cholesterol biosynthetic pathway. Mevalonolactone was administered to provide a constant supply of newly synthesized cholesterol to cholesterol 7 alpha-hydroxylase, in order to overcome any inhibitory effect of taurocholate on HMG-Coa reductase. Infusions were started 72 h after biliary diversion, and carried out for an additional 48 h. Complete biliary diversion resulted in an increase in C7 alpha H specific activity (510%), protein mass (550%), steady-state mRNA levels (1430%), and transcriptional activities (330%) as compared to control rats with intact enterohepatic circulations. When rats with biliary diversion were infused intraduodenally with taurocholate, the specific activities of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase activities decreased by 75% (P less than 0.001) and 73% (P less than 0.001), respectively. Cholesterol 7 alpha-hydroxylase mass, mRNA, and transcriptional activity decreased after intraduodenal infusion of taurocholate to levels similar to those of rats with an intact enterohepatic circulation. The combination of constant infusion of mevalonate and taurocholate failed to reverse the inhibitory effects of taurocholate on cholesterol 7 alpha-hydroxylase activity, mRNA levels, and in vitro transcriptional rates. These data provide evidence that taurocholate represses cholesterol 7 alpha-hydroxylase at the level of gene transcription, and not via down-regulation of HMG-CoA reductase. Infusion of mevalonate alone to biliary diverted rats did not alter cholesterol 7 alpha-hydroxylase activity or mRNA levels, while leading to a 57% decrease in C7 alpha H gene transcription. This latter finding suggests that mevalonate or its metabolites may be capable of stabilizing C7 alpha H mRNA levels while down-regulating transcriptional activity.

A proposal is made for a system of nomenclature of the more common unconjugated and conjugated bile acids. Acceptable trivial names for bile acids are tabulated, and guidelines are proposed for using these existing trivial names as roots to create acceptable semi-systematic names for other bile acids, as well as for new natural bile acids that will be discovered in the future. The term alpha-hyocholic is recommended to replace hyocholic, and beta-hyocholic to replace omega-muricholic. The term murideoxycholic acid is recommended for 3 alpha,6 beta-dihydroxy-5 beta-cholan-24-oic acid. Proposals are also made for bile acids with epimeric hydroxy groups, for unsaturated bile acids, and for bile acids with oxo- and/or hydroxy-oxo- substituents on the nucleus and/or on the side chain. For conjugated bile acids, the term "aminoacyl amidates" is recommended to replace "amidates" for bile acids conjugated in N-acyl linkage with amino acids. Nomenclature for other types of conjugates (sulfates, glucuronides, glucosides) is included as well as abbreviations. It is recommended that the historic tradition of naming a newly discovered bile acid after the species from which it was isolated be abandoned, and that in the future such a bile acid should be named using the principles contained in this paper.

Effects of expansion of the hepatic free cholesterol pool on bile acid and cholesterol metabolism and homeostasis were examined in rats fed cholesterol in high-fat diets or treated with oleyl-p-(n-decyl)-benzenesulfonate (ODS) or progesterone. Cholesterol feeding for 10-16 days, which increased free (33%) and esterified (6-fold) cholesterol, had no effect on cholate synthesis, total bile acid synthesis, or cholate turnover, whereas these activities were increased 60-80% by ODS and progesterone, which produced only small increases (19%) in free cholesterol. Cholesterol feeding reduced beta-hydroxy-beta-methylglutaryl (HMG)-CoA reductase (72%) and cholesteryl ester hydrolase (48%) and increased acyl-CoA:cholesterol acyltransferase (184%), whereas ODS and progesterone reversed these compensatory responses in cholesterol-fed rats. Cholesterol 7 alpha-hydroxylase was changed no more than 22% by any treatment. A bolus of ODS elevated biliary cholesterol output 41% and shifted biliary bile acid synthesis and composition toward 12-deoxy bile acids. These effects were not seen in ODS-fed or progesterone-treated rats, in which cholesteryl ester stores were depleted. It is concluded that effects of free cholesterol on bile acid synthesis and biliary cholesterol are probably mediated by specific precursor or regulatory pools which can be independently regulated and which represent a relatively small fraction of hepatic free cholesterol.

We have previously reported that relatively hydrophobic bile acids, decreased hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase (reductase) activity whereas, hydrophilic bile acids had little effect on the enzyme. The purpose of the present study was to determine in more detail the mechanism of down-regulation of hepatic reductase activity by hydrophobic bile salts. Groups of rats were fed bile acids of differing hydrophobicity: ursodeoxycholic, cholic (CA), chenodeoxycholic (CDCA), deoxycholic (DCA), or cholesterol for 14 days. Reductase specific activities and concentrations of reductase protein were determined in hepatic microsomes. Quantitation of "steady state" levels of reductase mRNA was performed using Northern and dot blot hybridization. Reductase gene transcriptional activity (nuclear "run-on") was determined in nuclei isolated from livers of animals fed different bile acids. Hydrophobic bile acids and cholesterol significantly decreased reductase activity: CA (57%), CDCA (77%), DCA (73%), cholesterol (89%), and reductase protein levels as measured by an enzyme-linked immunosorbent assay method were also decreased; CA (27%), CDCA (31%), DCA (42%), and cholesterol (35%). Reductase mRNA levels were also decreased after feeding hydrophobic bile acid: CA (43%), CDCA (47%), DCA (54%), and cholesterol (53%). Ursodeoxycholic, a hydrophilic bile acid, caused a much smaller decrease in reductase activity (18%), protein mass (16%), and mRNA levels (10%). Decreased transcriptional activities were observed in CA- and cholesterol-fed rats. Surprisingly, CDCA- and DCA-fed animals showed transcriptional activities similar to control animals even though steady state mRNA levels were low in CDCA- and DCA-fed animals. We hypothesize a post-transcriptional regulation of reductase mRNA by hydrophobic bile acids.

Cholesterol 7 alpha-hydroxylase catalyzes the rate-limiting step in the bile acid biosynthetic pathway. Regulation of this pathway is thought to occur solely as a result of a negative feedback control mechanism that is dependent upon the flux and composition of bile salts undergoing enterohepatic circulation. We have used the chronic biliary diverted (CBD) rat model to study the mechanism of regulation of cholesterol 7 alpha-hydroxylase by taurocholate. As compared to nonoperated controls, CBD rats exhibited a 4.2-fold increase in cholesterol 7 alpha-hydroxylase-specific activity, a 4.5-fold increase in enzyme mass, a 10-fold increase in steady-state mRNA levels, and a 3.4-fold increase in transcriptional (nuclear "run-on") activity. Intraduodenal infusion of taurocholate at a rate of 36 mumol/100 g/h for 48 h in CBD rats caused a significant (p less than 0.05) decrease (64%) in cholesterol 7 alpha-hydroxylase-specific activity, mass (72%), steady-state mRNA levels (74%), and transcriptional activity (57%) as compared to CBD controls. Cholesterol feeding increased cholesterol 7 alpha-hydroxylase-specific activity (288%), poly(A) RNA levels (291%), and transcriptional activity (220%) as compared to control animals. These results provide convincing evidence that bile salts, either directly or indirectly, down-regulate in vivo transcription of the cholesterol 7 alpha-hydroxylase gene, which is probably the major mechanism regulating the levels of this enzyme. The results of this study also suggest that the promoter for cholesterol 7 alpha-hydroxylase may have both bile salt- and sterol-responsive elements.

The role of hepatocytes in bile acid and cholesterol metabolism has been extensively studied. By contrast, nothing is known about the role of bile ductular epithelial cells in cholesterol and bile acid metabolism. The purpose of the current studies was to establish whether bile ductular epithelial cells synthesize cholesterol, bile acids or both and to determine whether these cells are capable of metabolizing (hydroxylating, conjugating) bile acids. Bile ductular epithelial cells were isolated from rat liver after ligation of the common bile duct for 6 to 8 wk. Bile ductular epithelial cells were essentially free (greater than 99%) of hepatocytes and were histochemically positive (greater than 80%) for gamma-glutamyl transpeptidase activity. Cholestatic hepatocytes were simultaneously isolated and characterized with regard to their ability to synthesize and metabolize bile acids. Incubation of bile ductular epithelial cells with [14C]-acetate resulted in rapid labeling of cellular cholesterol, suggesting that these cells have a complete cholesterol biosynthetic pathway. The addition of [4-14C]-cholesterol to bile ductular epithelial cells did not lead to detectable synthesis of [14C]-bile acids. [24-14C]-Cholic acid, [24-14C]-deoxycholic acid, [24-14C]-lithocholic acid and [3H]-ursodeoxycholic acid were individually added to bile ductular epithelial cells and incubated for 24 or 48 hr. Bile acid metabolites were extracted and separated by C-18 reverse-phase high-performance liquid chromatography or thin-layer chromatography. Bile ductular epithelial cells conjugated deoxycholic acid, ursodeoxycholic acid and lithocholic acid to glycine and taurine. Surprisingly, no conjugation of cholic acid was detected. Conjugated lithocholic acid was further metabolized to highly polar metabolite(s), possibly beta-muricholic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol, despite its poor solubility in aqueous solutions, exchanges efficiently between membranes. Movement of cholesterol between different subcellular membranes in the hepatocyte is necessary for assembly of lipoproteins, biliary cholesterol secretion, and bile acid synthesis. Factors which initiate and facilitate transfer of cholesterol between different membranes in the hepatocyte are incompletely understood. It is known that cholesterol secretion into the bile is linked to bile salt secretion. In the present study, we investigated the effects of bile salts of different physicochemical properties at submicellar concentrations (150- 600 microM) on the transfer of [14C]cholesterol from hepatocytes, or crude hepatocellular membranes (donors), to rat high density lipoproteins (acceptor). Bile salts included taurine conjugates of ursodeoxycholic acid (TUDCA), hyodeoxycholic acid (THDCA), cholic acid (TCA), chenodeoxycholic acid (TCDCA), and deoxycholic acid (TDCA). High density lipoprotein (HDL) was separated from hepatocellular membranes and the transfer of [14C]cholesterol from the membranes to HDL was quantitatively determined. In the absence of HDL, [14C]cholesterol remained confined to the membrane fraction. Following addition of HDL, [4-14C]cholesterol in the HDL fraction increased linearly over time. Addition of hydrophilic bile salts (TUDCA and THDCA) increased transfer of [4-14C]cholesterol to HDL only minimally. By contrast, more hydrophobic bile salts stimulated transfer of labeled cholesterol to HDL, and their potency increased in order of increasing hydrophobicity (TCA less than TCDCA less than TDCA). Both for single bile salts and mixtures of bile salts at a total bile salt concentration of 0.30 mM, the rate of cholesterol transfer exhibited a strong linear correlation with a bile salt monomeric hydrophobicity index (r = 0.95; P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro and in vivo studies have shown that the sterol 27-hydroxylase (CYP27) gene is transcriptionally repressed by hydrophobic bile acids. The molecular mechanism(s) of repression of CYP27 by bile acids is unknown. To identify the bile acid responsive element (BARE) and transcription factor(s) that mediate the repression of CYP27 by bile acids, constructs of the CYP27 5'-flanking DNA were linked to either the CAT or luciferase reporter gene and transiently transfected into primary rat hepatocytes. Taurocholate (TCA), taurodeoxycholate (TDCA) and taurochenodeoxycholate (TCDCA) significantly reduced CAT activities of the -840/+23, -329/+23, and -195/+23 mCAT constructs. A -76/+23 construct showed no regulation by bile acids. When a DNA fragment (-110/-86) from this region was cloned in front of an SV 40 promoter it showed down-regulation by TDCA. 'Super'-electrophoretic mobility shift assays (EMSA) indicated that both HNF1alpha and C/EBP bind to the -110 to -86 bp DNA fragment. Recombinant rat HNF1alpha and C/EBPalpha competitively bound to this DNA fragment. 'Super'-EMSA showed that TDCA addition to hepatocytes in culture decreased HNF1alpha, but not C/EBP, binding to the -110/-86 bp DNA fragment. A four base pair substitution mutation (-103 to -99) in this sequence eliminated TCA and TDCA regulation of the (-840/+23) construct. The substitution mutation also eliminated (95%) HNF1alpha, but not C/EBP, binding to this DNA fragment. We conclude that bile acids repress CYP27 transcription through a putative BARE located between -110 and -86 bp of the CYP27 promoter. The data suggest that bile acids repress CYP27 transcriptional activity by decreasing HNF1alpha binding to the CYP27 promoter.

The feedback repression of cholesterol 7alpha-hydroxylase transcriptional activity and mRNA levels by taurocholate (TCA) occurs via a protein kinase C (PKC)-dependent signal. To determine whether bile acids could activate PKC indirectly via generation of diacylglycerol (DG), their effects on DG levels in primary cultures of rat hepatocytes were determined using a DG kinase assay. To determine whether bile acids might activate PKC isozymes more directly, their effects on PKC alpha and delta purified from baculovirus expression systems were examined in phosphatidylserine/phosphatidylcholine/Triton X-100 (PS/PC/TX) mixed micelles. Addition of tauroursodeoxycholate (TUDCA), taurocholate (TCA), or taurodeoxycholate (TDCA) (50 microM) to the cells rapidly (15 min) increased DG content in cultured rat hepatocytes to 105%, 155%, and 130%, respectively, as compared to untreated control cultures. Addition of TCA increased PKC alpha specific activity with EC50 of approximately 400 nM; maximal activity was observed with 5 microM. Other taurine-conjugated bile acids (5 microM) increased PKC alpha specific activity (pmol/min/microg protein) in proportion to their relative hydrophobicity: PS/PC/TX 17 +/- 2; + TUDCA 29 +/- 18; + TCA 68 +/-13; + TDCA 166 +/- 21; and, taurochenodeoxycholate 178 +/- 20 (P vs. PS/PC/TX = 0.54, 0.019, 0.002, and 0.001, respectively); unconjugated bile acids gave similar results (r2 for activity vs. hydrophobicity index 0.59). Taurine-conjugated bile acid interaction enthalpies, as determined by dimyristoyl-phosphatidylcholine chromatography, were more highly correlated (r2 = 0.96) with PKC alpha activation than with the hydrophobicity index. TCA also stimulated the activity of purified PKCdelta with EC50 of approximately 150 nM and maximally (2.7-fold) at 1 microM. Free and taurine-conjugated bile acids (1 microM) increased PKCdelta activity according to their hydrophobicity index (r2 = 0.89) and interaction enthalpies (r2 = 0.96).

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. (Gastroenterology 1997 Dec;113(6):1949-57)

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.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.Transporter protein and mRNA levels in cholic acid-fed rats increased approximately threefold above levels in paired rats fed the control diet (P0.02). 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 (P0.0001). The intraduodenal infusion of taurocholate (24 hours) in biliary-diverted rats resulted in a time-dependent reinduction of transporter protein expression (3.5-fold).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.

Cholesterol degradation to bile acids represents 50% of total elimination of cholesterol from the body each day. Cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase catalyze initial steps in the neutral and acidic pathways, respectively. Both enzymes were recently cloned and sequenced, and hence the molecular basis of their regulation could be studied. In the rat, cholesterol 7 alpha-hydroxylase is regulated by three classes of effector molecules: a) hydrophobic (but not hydrophilic) bile acids, b) cholesterol, and c) hormones (glucocorticoids plus thyroxine, glucagon and insulin). Most studies presented so far indicate that regulation of cholesterol 7 alpha-hydroxylase probably occurs at the level of gene transcription. Sterol 27-hydroxylase, a mitochondrial P-450 enzyme, appears to be regulated by hydrophobic bile acids, but to a lesser extent than cholesterol 7 alpha-hydroxylase. The contribution of this acidic pathway to total bile acid synthesis is not known but it appears to be more significant than previously thought.

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.

Cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in the bile acid synthesis pathway, is down-regulated by taurocholate by way of negative feedback control at the level of gene transcription. The molecular basis of regulation of cholesterol 7 alpha-hydroxylase by other hydrophobic bile salts and under more physiological conditions is not known. The aim of this study was to investigate the molecular basis of regulation of cholesterol 7 alpha-hydroxylase by several naturally occurring bile salts in rats with intact enterohepatic circulation. Male Sprague-Dawley rats were pair-fed for 14 days normal chow (control), cholestyramine (5% of diet), cholic acid (1%), chenodeoxycholic acid (1%) or deoxycholic acid (0.25%). When rats were killed, livers were harvested and HMG-CoA reductase specific activity and cholesterol 7 alpha-hydroxylase specific activities, steady-state mRNA levels and transcriptional activity were determined and compared with those of control rats fed normal chow. Compared with results in paired controls, cholestyramine feeding led to an approximate threefold increase in HMG-CoA reductase specific activity. Feeding of hydrophobic bile salts profoundly decreased the specific activity of HMG-CoA reductase. Cholestyramine led to a three-fold increase in cholesterol 7 alpha-hydroxylase specific activity, steady-state mRNA levels and gene transcriptional activity. The feeding of cholic (1%), chenodeoxycholic (1%) and deoxycholic acid (0.25%) led to significant decreases in cholesterol 7 alpha-hydroxylase specific activities (62%, 84% and 97%, respectively), steady-state mRNA levels (72%, 29% and 61%, respectively) and transcriptional activities (44%, 43% and 54%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in the bile acid biosynthetic pathway, is thought to be regulated by hydrophobic bile acids through negative feedback control. The role of cholesterol in the regulation of cholesterol 7 alpha-hydroxylase is more controversial, in part because of incomplete understanding of the relationship between the pathways of cholesterol synthesis and degradation. The main objective of this study was to define the interaction between these two pathways in an experimental model in which the supply of newly synthesized cholesterol was interrupted by sustained infusion of mevinolin (lovastatin), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) or accelerated by a continuous infusion of mevalonate, a cholesterol precursor. The study was carried out in rats subjected to short-term bile fistula. In one set of experiments, rats were treated postoperatively with mevinolin (5 mg/kg loading dose followed by 2 mg/kg/hr infusion), mevalonate (180 mumol/hr infusion) or both for up to 96 hr. In a separate set of experiments, rats were infused intraduodenally with taurocholate (36 mumol/100 gm/hr for up to 96 hr). We determined cholesterol 7 alpha-hydroxylase- and HMG-CoA reductase specific activities at those time intervals, whereas bile acid synthesis rates were determined throughout the study. Compared with rats not subjected to surgery, rats with short-term biliary diversion had increases in cholesterol 7 alpha-hydroxylase activity of 259% and 827% at 48 and 96 hr, respectively. The increase in bile acid biosynthesis was less pronounced. Continuous infusion of mevinolin completely prevented increases in cholesterol 7 alpha-hydroxylase specific activity and bile acid biosynthesis at both time intervals.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of bile acids in the regulation of cholesterol 7 alpha-monooxygenase (EC 1.14.13.17) was characterized using primary cultures of rat hepatocytes supplemented with dexamethasone and thyroxine. Taurocholate and taurodeoxycholate (50 microM) repressed cholesterol 7 alpha-hydroxylase mRNA to 44 +/- 9 and 52 +/- 4%, respectively, of control values. Repression by these natural, relatively hydrophobic bile acids was concentration dependent, with an IC50 of about 50 microM, and time dependent with a t1/2 for repression of 22 h. In contrast, two natural hydrophilic bile acids, tauroursodeoxycholate and taurohyodeoxycholate, had no effect on cholesterol 7 alpha-hydroxylase mRNA levels. Taurochenodeoxycholate and taurolithocholate also had no effect, but these hydrophobic bile acids were rapidly hydroxylated to more hydrophilic bile acids. Hydrophilic bile acid analogues (nor (C23) bile acids and beta-hydroxy epimers) repressed cholesterol 7 alpha-hydroxylase mRNA less potently than their corresponding and more hydrophobic C24 or alpha-hydroxy derivatives. Cholesterol 7 alpha-hydroxylase specific activity was decreased by taurocholate or taurodeoxycholate (50 microM) to 26 +/- 9 and 56 +/- 3% of control, respectively; its transcriptional activity was repressed to 52 +/- 5% of control by taurocholate (50 microM). The addition of cholesterol or the induction of cholesterol biosynthesis did not influence repression of cholesterol 7 alpha-hydroxylase mRNA levels by taurocholate. Based on several lines of evidence, cAMP was not involved in bile acid-induced repression. In rat hepatocytes cultured under conditions in which cholesterol 7 alpha-hydroxylase gene expression is maintained at in vivo levels, hydrophobic bile acids repress this enzyme at the level of gene transcription independently of cholesterol availability.

In primary cultures of adult rat hepatocytes the level of cholesterol 7 alpha-hydroxylase steady-state mRNA markedly decreased by 72 h. However, the addition of L-thyroxine (T4) and dexamethasone synergistically returned cholesterol 7 alpha-hydroxylase steady-state mRNA levels near to that of cholestyramine-fed animals. The maximal responses to T4 and dexamethasone in serum-free medium were at 1.0 and 0.1 microM, respectively. The addition of T4 in combination with dexamethasone resulted in an 11-fold increase in transcriptional activity of the cholesterol 7 alpha-hydroxylase gene as compared to no addition controls. The specific activities of cholesterol 7 alpha-hydroxylase in microsomes prepared from cultures treated with dexamethasone and T4 were 1.56 +/- 1.17 nmol/h/mg protein which is similar to that of intact liver (1.70 +/- 0.062 nmol/h/mg protein), but lower than cholestyramine-fed animals. Cholesterol 7 alpha-hydroxylase activity was not detectable (less than 0.020 nmol/h/mg protein) at 72 h in cultures without the addition of both dexamethasone and T4. In the presence of optimal concentrations of dexamethasone and T4, glucagon (0.2 microM), or dibutyryl cAMP (50 microM) decreased (90%) cholesterol 7 alpha-hydroxylase mRNA within 6 h. Transcriptional activity decreased (62%) in 6 h following the addition of glucagon (0.2 microM) to the culture medium. The results reported in this paper suggest an important role for multiple hormones in the regulation of cholesterol 7 alpha-hydroxylase in the liver.

We have previously reported that relatively hydrophobic bile acids, decreased hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase (reductase) activity whereas, hydrophilic bile acids had little effect on the enzyme. The purpose of the present study was to determine in more detail the mechanism of down-regulation of hepatic reductase activity by hydrophobic bile salts. Groups of rats were fed bile acids of differing hydrophobicity: ursodeoxycholic, cholic (CA), chenodeoxycholic (CDCA), deoxycholic (DCA), or cholesterol for 14 days. Reductase specific activities and concentrations of reductase protein were determined in hepatic microsomes. Quantitation of "steady state" levels of reductase mRNA was performed using Northern and dot blot hybridization. Reductase gene transcriptional activity (nuclear "run-on") was determined in nuclei isolated from livers of animals fed different bile acids. Hydrophobic bile acids and cholesterol significantly decreased reductase activity: CA (57%), CDCA (77%), DCA (73%), cholesterol (89%), and reductase protein levels as measured by an enzyme-linked immunosorbent assay method were also decreased; CA (27%), CDCA (31%), DCA (42%), and cholesterol (35%). Reductase mRNA levels were also decreased after feeding hydrophobic bile acid: CA (43%), CDCA (47%), DCA (54%), and cholesterol (53%). Ursodeoxycholic, a hydrophilic bile acid, caused a much smaller decrease in reductase activity (18%), protein mass (16%), and mRNA levels (10%). Decreased transcriptional activities were observed in CA- and cholesterol-fed rats. Surprisingly, CDCA- and DCA-fed animals showed transcriptional activities similar to control animals even though steady state mRNA levels were low in CDCA- and DCA-fed animals. We hypothesize a post-transcriptional regulation of reductase mRNA by hydrophobic bile acids.

Cholesterol 7 alpha-hydroxylase catalyzes the rate-limiting step in the bile acid biosynthetic pathway. Regulation of this pathway is thought to occur solely as a result of a negative feedback control mechanism that is dependent upon the flux and composition of bile salts undergoing enterohepatic circulation. We have used the chronic biliary diverted (CBD) rat model to study the mechanism of regulation of cholesterol 7 alpha-hydroxylase by taurocholate. As compared to nonoperated controls, CBD rats exhibited a 4.2-fold increase in cholesterol 7 alpha-hydroxylase-specific activity, a 4.5-fold increase in enzyme mass, a 10-fold increase in steady-state mRNA levels, and a 3.4-fold increase in transcriptional (nuclear "run-on") activity. Intraduodenal infusion of taurocholate at a rate of 36 mumol/100 g/h for 48 h in CBD rats caused a significant (p less than 0.05) decrease (64%) in cholesterol 7 alpha-hydroxylase-specific activity, mass (72%), steady-state mRNA levels (74%), and transcriptional activity (57%) as compared to CBD controls. Cholesterol feeding increased cholesterol 7 alpha-hydroxylase-specific activity (288%), poly(A) RNA levels (291%), and transcriptional activity (220%) as compared to control animals. These results provide convincing evidence that bile salts, either directly or indirectly, down-regulate in vivo transcription of the cholesterol 7 alpha-hydroxylase gene, which is probably the major mechanism regulating the levels of this enzyme. The results of this study also suggest that the promoter for cholesterol 7 alpha-hydroxylase may have both bile salt- and sterol-responsive elements.

The protective effect of ursodeoxycholate conjugates against bile salt hepatotoxicity was studied in chronic bile fistula rats. Taurochenodeoxycholate or taurodeoxycholate, infused intraduodenally at 24 or 16 mumols/100 g rat per hour, respectively, caused cholestasis and severe hepatocellular necrosis within 8 hours. In contrast, tauroursodeoxycholate or taurocholate at 48 mumols/100 g rat per hour were choleretic. Tauroursodeoxycholate was not hepatotoxic, whereas taurocholate produced moderate hepatocellular necrosis. Simultaneous infusion of tauroursodeoxycholate to rats receiving taurochenoxycholate or taurodeoxycholate preserved bile flow and ameliorated hepatic injury in a dose-dependent manner. Tauroursodeoxycholate protected equally by intravenous and intraduodenal routes. Intravenous glycoursodeoxycholate also was protective. The hydrophobicity index of infused bile salts correlated well with their toxicity. Concurrent administration of ursodeoxycholate conjugates did not reduce biliary recovery of intraduodenally infused [24-14C]-taurocholate. Biliary alkaline phosphatase secretion was stimulated by infusion of taurocholate, taurodeoxycholate, or taurochenodeoxycholate; simultaneous infusion of ursodeoxycholate conjugates failed to prevent this increase. We conclude that ursodeoxycholate counteracts hepatoxicity of more hydrophobic bile salts via a direct effect at the level of the liver.

In the chronic bile fistula rat, the administration of a bolus dose of mevinolinic acid, an inhibitor of HMG-CoA reductase, was followed by rapid down-regulation of cholesterol 7 alpha-hydroxylase activity and a decrease in bile acid synthesis. These observations suggested that either newly synthesized cholesterol or some other metabolite of mevalonate may be involved in the regulation of bile acid synthesis. In order to distinguish between these two alternatives, we carried out experiments in which cholesterol synthesis was blocked by AY9944, a compound that inhibits the conversion of 7-dehydrocholesterol to cholesterol, a last step in the cholesterol biosynthesis pathway. Rats underwent biliary diversion for 72 h at which time they were given intravenously either a bolus dose of AY9944 (1 mg/kg) or control vehicle. At 0 (pre-treatment control), 0.5, 1.5, and 3 h post bolus, livers were harvested and specific activities of cholesterol 7 alpha-hydroxylase were determined. At 1.5, 3, and 6 h post bolus, AY9944 inhibited bile acid synthesis by 19 +/- 6%, 40 +/- 4%, and 41 +/- 6%, respectively, as compared to pretreatment baseline. Cholesterol 7 alpha-hydroxylase activity determined at 0.5, 1.5, and 3 h was decreased by 44 +/- 6%, 44 +/- 2%, and 36 +/- 2%, respectively, as compared to the control value. In in vitro experiments using microsomes from livers of control bile fistula rats, the addition of AY9944 (up to 100 microM) failed to inhibit cholesterol 7 alpha-hydroxylase activity. The results of this study demonstrate that, in the chronic bile fistula rat, acute inhibition of cholesterol synthesis at either early or late steps leads to a rapid down-regulation of cholesterol 7 alpha-hydroxylase activity and decrease in bile acid synthesis.

Under most experimental conditions, the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase) and cholesterol 7 alpha-hydroxylase, change together in parallel directions. It has been suggested that newly synthesized cholesterol may be the preferred substrate for cholesterol 7 alpha-hydroxylase, which may account for the observed synchronous behavior of the two enzymes. To test this hypothesis, mevinolinic acid, a potent competitive inhibitor of HMG-CoA reductase, was administered as a single intravenous bolus (10 mg/kg) to rats with a chronic bile fistula. Bile acid synthesis was determined following inhibition of HMG-CoA reductase by mevinolinic acid over a 27-h time course and specific activities of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase were determined in liver microsomes. At 3, 6, and 27 h after a bolus dose of mevinolinic acid, bile acid synthesis was reduced by 54 +/- 5%, 42 +/- 8%, and 23 +/- 13%, respectively, from preinfusion baseline. Within 30 min after administration of mevinolinic acid, HMG-CoA reductase activity was inhibited by at least 87%. At 0.5, 1.5, 3, 6, and 27 h after mevinolinic acid injection, cholesterol 7 alpha-hydroxylase activity was decreased by 6%, 25%, 54%, 41%, and 17%, respectively. By 27 h, the activities of both enzymes had returned to baseline levels. The reduction of bile acid synthesis correlated closely with the observed changes in the activities of cholesterol 7 alpha-hydroxylase. In vitro addition of mevinolinic acid (up to 20 microM) to rat liver microsomes failed to inhibit cholesterol 7 alpha-hydroxylase activity, suggesting no direct effect of mevinolinic acid on enzyme activity. When a bolus dose of mevinolinic acid was coupled with a continuous infusion of mevalonate, the product of the reaction catalyzed by HMG-CoA reductase, the mevinolinic acid-induced decrease in cholesterol 7 alpha-hydroxylase activity and bile acid synthesis was prevented. The results of this study provide evidence that, under the experimental conditions described, there is a linkage between the rates of cholesterol synthesis and the activities of cholesterol 7 alpha-hydroxylase. The data also emphasize the importance of the newly synthesized cholesterol in the regulation of cholesterol 7 alpha-hydroxylase activity.

Monolayer cultures of hepatocytes isolated from cholestyramine-fed rats and incubated in serum-free medium converted exogenous [4-14C]cholesterol into bile acids at a 3-fold greater rate than did cultures of hepatocytes prepared from untreated rats. Cholic acid and beta-muricholic acid identified and quantitated by gas-liquid chromatography and thin-layer chromatography were synthesized by cultured cells for at least 96 h following plating. The calculated synthesis rate of total bile acids by hepatocytes prepared from cholestyramine-fed animals was approximately 0.058 micrograms/mg protein/h. beta-Muricholic acid was synthesized at approximately a 3-fold greater rate than cholic acid in these cultures. Cultured hepatocytes rapidly converted the following intermediates of the bile acid pathway; 7 alpha-hydroxy[7 beta-3H]cholesterol, 7 alpha-hydroxy-4-[6 beta-3H] cholesten-3-one, and 5 beta-[7 beta-3H]cholestane-3 alpha, 7 alpha, 12 alpha-triol into bile acids. [24-14C]Chenodeoxycholic acid and [3H]ursodeoxycholic acid were rapidly biotransformed to beta-muricholic acid. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity measured in microsomes of cultured hepatocytes decreased during the initial 48 h following plating, but remained relatively constant for the next 72 h. In contrast, cholesterol 7 alpha-hydroxylase activity appeared to decrease during the first 48 h, followed by an increase over the next 48 h. Despite the apparent changes in enzyme activity in vitro, the rate of bile acid synthesis by whole cells during this time period remained constant. It is concluded that primary monolayer cultures of rat hepatocytes can serve as a useful model for studying the interrelationship between cholesterol and bile acid metabolism.

The present report has presented the first clear evidence in man for the existence of specific hepatic cholesterol precursor sites associated with the formation and secretion of bile acids and biliary cholesterol. These hepatic compartments derive virtually all their cholesterol from newly synthesized and lipoprotein free cholesterol. The model which is presented was formulated on current concepts of cholesterol metabolism in man and is concerned, at this initial stage, with the elucidation of the bile acid and biliary cholesterol compartments. The complexity of cholesterol metabolism in man necessitated an initial approach that would minimize the number of inputs of cholesterol into the system, allow for the sampling of several cholesterol compartments, and permit the simultaneous labeling of newly synthesized cholesterol and preformed cholesterol. To achieve these objectives, we studied the patient with a total bile fistula. Six patients were administered simultaneously pulse injections of labeled mevalonic acid and [14C]cholesterol. The qualitative features of the specific activity time course curves after labeled mevalonic acid revealed no precursor-product relationship between bile acid, biliary cholesterol, and plasma free cholesterol. The peak specific activity of the bile acids was reached in approximately 100 min and was higher than the biliary cholesterol, which was higher than the plasma free cholesterol. The plasma free cholesterol specific activity became higher than the other lipids after 12 h and remained higher throughout the period of study. Similar related observations were made with [14C]cholesterol. The data were then subjected to simulation analysis and modeling using the SAAM-27 computer program. Computer least-square fits of the data were obtained after the model was evolved. During the model development, the least number of compartments and transport pathways were introduced consistent with a good fit of the data. Of particular importance was the constraint that the model fit the data obtained from both [14C]cholesterol and labeled mevalonic acid. The same parameter values were used to fit the data from both tracers. The fluxes arrived at in the model indicate that 31% and 20%, respectively, of the cholesterol input into the bile acid and biliary cholesterol precursor sites were derived directly from the newly synthesized hepatic cholesterol. The remainder had its origin predominantly from lipoprotein free cholesterol. Plasma esterified cholesterol (as free) made a small contribution (11%) to the bile acid compartment. Similarly, 10% of the biliary cholesterol arose from an unknown hepatic site. The present report has provided the basis for a new procedure for studying in vivo cholesterol metabolism in man. Examination of the derived cholesterol flux rates between the compartments suggests the presence of an important mechanism regulating the partitioning of lipoprotein free cholesterol between the bile acid and biliary cholesterol precursor sites. Aberrations in the proportioning of precursor cholesterol between these sites could be a causative factor precipitating the excessive secretion of biliary cholesterol and the production of lithogenic bile.

The role of bile canalicular and microsomal membranes in the synthesis and transport of biliary lipids was investigated by using the isolated perfused rat liver model. Labeled lecithin precursors ((3H)-palmitic acid, (14C)linoleic acid, (3H)choline, and 32PO4) and a cholesterol precursor ((3H)mevalonic acid) were administered with and without sodium taurocholate. The incorporation pattern of these labeled precursors into linoleyl and arachidonyl lecithins and cholesterol fractions of microsomes, bile canaliculi, and bile were examined at 30-min intervals up to 90 min. Marker enzymes and electron microscopy indicated that isolated subfractions of plasma membranes were enriched with bile canaliculi (less than 10 percent microsomal contamination). Taurocholate significantly stimulated the incorporation of 32PO4, (3H)choline, (3H)palmitic acid, and (14C)linoleic acid into linoleyl and arachidonyl lecithin with parallel incorporation curves for microsomal and bile canalicular membranes throughout the 90-min study period. During the 30-60-min period, however, these same lecithin fractions in bile significantly exceeded the specific activity of the membrane lecithins. The enzyme CDP-choline diglyceride transferase was virtually absent from canaliculi relative to microsomes, indicating that canaliculi lack the capacity for de novo lecithin synthesis. Incorporation of (3H)mevalonic acid into membranous and biliary cholesterol followed a pattern similar to that for lecithin. These data provide evidence that (a) biliary lecithin and cholesterol are derived from a microsomal subpool regulated by the flux of enterohepatic bile acids, (b) the role of the bile canalicular membranes with respect to biliary lipids is primarily transport rather than synthesis, and (c) lecithin and cholesterol are transported together from microsomes to bile. The findings are consistent with the existence of a cytoplasmic lipid complex within the hepatocyte which is actively involved in the intermembrane transport of biliary lipid.

The gastric secretory response to prompt intravenous injection of pentagastrin was investigated in 3 normal subjects and 3 patients with duodenal ulcer. The highest responses to pentagastrin and histamine were correlated. Intravenous pentagastrin in a dose of 0.5 μg per kg could thus achieve the same extent of discrimination between individuals with a 10-fold economy in dosage over subcutaneous pentagastrin. Sensitivity to pentagastrin, expressed in the D50, was higher in patients with duodenal ulcer. Unexpectedly, the highest response to intravenous pentagastrin was only 40% of the peak response to subcutaneous histamine. Accordingly, intravenous pentagastrin acted as a partial agonist with an efficacy of about 0.4. The effect of prompt injection of pentagastrin contrasted with the effect of slow intravenous infusion of pentagastrin, as well as with the effects of gastrin-17 given by either prompt injection or slow infusion. A model is proposed to account for these and related findings in other species. The secretory receptor for gastrin and its analogues is viewed as consisting of two sites: a high affinity stimulatory site and a low affinity inhibitory site. Partial agonism after prompt intravenous injection would result from blood and tissue transients which are successively too high (partially inhibitory) and low (stimulatory). The disparate effects of analogues and the differences between species would be determined by the relative affinities of the two sites. The overflow of stimulant into inhibitory sites would account for the reversal of response at high doses observed in numerous studies.

The in vivo conversion of several 5 beta-cholestane intermediates to primary bile acids was investigated in three patients with total biliary diversion. The following compounds were administered intravenously: 5 beta-[G-3H]-cholestane-3 alpha, 7 alpha-diol, 5 beta-[G-3H]cholestane-3 alpha, 7alpha, 26-triol, and 5 beta-[24-14C]cholestane-3 alpha, 7 alpha-25-triol. Bile was then collected quantitatively at frequent intervals for the next 21 to 28 h. The administered 5 beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol was found to be efficiently converted to cholic and chenodeoxycholic acids in two patients; 61 and 75% of the administered label was found in primary bile acids. The proportion of labeled cholic to chenodeoxycholic acid was 1.20 and 1.02 in the bile of these patients, indicating that the C-26 triol was efficiently converted to cholic acid. The ratio of cholic to chenodeoxycholic acid (mass) in the bile of these patients was 1.23 and 2.32. The 5 beta-cholestane-3alpha, 7alpha-diol intermediate was also efficiently converted (71%) to both primary bile acids. The cholic to chenodeoxycholic acid ratios by mass and label were similar (2.97 versus 2.23). By contrast, the 5beta-cholestane-3alpha, 7alpha, 25-triol was poorly converted to bile acids in three patients. Following the administration of this compound almost all of the administered radioactivity found in the bile acid fraction was in cholic acid (5 to 19%) and very little (less than 5%) was found in chenodeoxycholic acid. These findings indicate that ring hydroxylation at position 12 is not materially hindered by the presence of a hydroxyl group on the side chain at C-26 in patients with biliary diversion. The labeled C-26-triol which was efficiently converted to both primary bile acids in a proportion similar to that which was observed for the bile acids synthesized by the liver suggests that this 5beta-cholestane derivative may be a major intermediate in the synthesis of both cholic and chenodeoxycholic acids.

The effect of individual 7 beta-hydroxy bile acids (ursodeoxycholic and ursocholic acid), bile acid analogues of ursodeoxycholic acid, combination of bile acids (taurochenodeoxycholate and taurocholate), and mixtures of bile acids, phospholipids and cholesterol in proportions found in rat bile, on bile acids synthesis was studied in cultured rat hepatocytes. Individual steroids tested included ursodeoxycholate (UDCA), ursocholate (UCA), glycoursodeoxycholate (GUDCA) and tauroursodeoxycholate (TUDCA). Analogues of UDCA (7-methylursodeoxycholate, sarcosylursodeoxycholate and ursooxazoline) and allochenodeoxycholate, a representative of 5 alpha-cholanoic bile acid were also tested in order to determine the specificity of the bile acid biofeedback. Each individual steroid was added to the culture media at concentrations ranging from 10 to 200 microM. Mixtures of taurochenodeoxycholate (TDCA) and taurocholate in concentrations ranging from 150 to 600 microM alone and in combination with phosphatidylcholine (10-125 microM) and cholesterol (3-13 microM) were also tested for their effects on bile acid synthesis. Rates of bile acid synthesis were determined as the conversion of added lipoprotein [4-14C]cholesterol or [2-14C]mevalonate into 14C-labeled bile acids and by GLC quantitation of bile acids secreted into the culture media. Individual bile acids, bile acid analogues, combination of bile acids and mixture of bile acids with phosphatidylcholine and cholesterol failed to inhibit bile acid synthesis in cultured hepatocytes. The addition of UDCA or UCA to the culture medium resulted in a marked increase in the intracellular level of both bile acids, and in the case of UDCA there was a 4-fold increase in beta-muricholate. These results demonstrate effective uptake and metabolism of these bile acids by the rat hepatocytes. UDCA, UCA, TUDCA and GUDCA also failed to inhibit cholesterol-7 alpha-hydroxylase activity in microsomes prepared from cholestyramine-fed rats. The current data confirm and extend our previous observations that, under conditions employed, neither single bile acid nor a mixture of bile acids with or without phosphatidylcholine and cholesterol inhibits bile acid synthesis in primary rat hepatocyte cultures. We postulate that mechanisms other than a direct effect of bile acids on cholesterol-7 alpha-hydroxylase might play a role in the regulation of bile acid synthesis.

A randomized double blind clinical comparison of neomycin and lactulose was performed in 33 cirrhotic patients with chronic portal-systemic encephalopathy (PSE) at seven cooperating hospitals. In order to maintain double blindness, sorbitol syrup was used as a control solution along with neomycin and was compared with lactulose syrup and placebo tablets in a double drug protocol. Twenty-nine patients were studied in a crossover investigation in which each received both therapeutic regimens preceded and followed by control periods. Four additional patients received one or the other agent, but did not recieve both. Serial, semiquantitative assessments were made in all patients of mental status, asterixis, and the trailmaking test (TMT) and electroencephalograms (EEG) and arterial ammonia levels. Both neomycin-sorbitol and lactulose were effective in the majority of patients (83 and 90%, respectively). Each of these parameters (mental state, asterixis, TMT, EEG, and NH3) was improved significantly by neomycin-sorbitol and lactulose. The post-treatment levels for each of these measures were similar in the neomycin and lactulose-treated groups. Mean stool pH was reduced by neomycinsorbitol to 6.1 and by lactulose to 5.5. This difference was highly significant statistically. Bowel activity was similar in the two groups. Both drugs were free of toxicity. These investigations demonstrate that both lactulose and neomycin-sorbitol are effective in the treatment of chronic portal-systemic encephalopathy.

Labeled cholic acid was administered orally to 8 patients with cirrhosis and no ascites. Bile-rich duodenal fluid was obtained at daily intervals for periods up to 7 days, and bile acid composition and cholic acid specific activity were determined. The total and component bile acid pools, cholic acid production, and half-life were estimated from the data and compared with a group of our patients without liver disease previously reported. The total and component bile acid pools were markedly reduced in patients with cirrhosis. The total bile acid pool was 46% smaller in cirrhotic patients than the control patients (1.3 g versus 2.4 g). There was a virtual absence of biliary deoxycholic acid in 7 of 8 patients with cirrhosis. Cholic acid production in the cirrhotic patients was approximately one-third that of the normal group. A prolongation of the cholic acid turnover time was also noted in 3 of the 8 cirrhotic patients. It is suggested that in patients with cirrhosis the failure of the liver to produce adequate amounts of bile acids is responsible for the diminished bile acid pool. The nonpancreatogenic steatorrhea observed in some patients with cirrhosis might be attributed in part to a lack of sufficient bile salts in the intestine.

Gallbladder bile, hepatic bile, and gallstones were obtained from 20 patients at surgery. The bile was analyzed for bile acids, cholesterol, and phospholipids and the gallstones for cholesterol. There was no significant difference in the lipid composition between the gallbladder and hepatic bile of patients with cholesterol gallstones. The gallbladder and hepatic bile of patients with cholesterol gallstones was out of the micellar zone or near the line of cholesterol saturation. Four patients who had pigmented stones with only traces of cholesterol had normal hepatic and gallbladder bile. Two patients had no demonstrable gallstones, but the gallbladder and hepatic bile were abnormal. The results of the present study suggest that the gallbladder does not play a major role in the alteration of the bile secreted by the liver and that the liver is the principal site for the production of the abnormal bile associated with cholesterol gallstone formation in man.