Diabetes and intestinal cholesterol uptake from bile salt solutions

A previously validated in vitro technique was used to determine the effect of diabetes mellitus on the intestinal uptake of cholesterol from various micellar bile salt solutions. The bile salts studied included cholic (C), taurocholic (TC), glycocolic (GC), chenodeoxycholic (CDC), taurochenodeoxycholic (TCDC), glycochenodeoxycholic (GCDC), deoxycholic (DC), taurodeoxycholic (TDC), and glycodeoxycholic (GDC). In control rats there was a reciprocal decline in cholesterol uptake with increasing concentrations of these nine bile acids, and cholesterol uptake was greater from the conjugated primary bile acids than from the unconjugated ones. With a 5 mM concentration of bile acids, the ratios of the uptake of 0.2 mM cholesterol in control rats were C = CDC = DC, TCDC greater than TC greater than TDC, and GC = GCDC greater than GDC; with 20 mM concentrations, the ratios of cholesterol uptake in control rats were C greater than CDC greater than DC, TC greater than TCDC greater than TDC, and GC = GCDC greater than GDC. In the diabetic animals cholesterol uptake was higher than in control rats when using 5 or 20 mM of each of the conjugated bile acids and with cholic acid. In contrast, cholesterol uptake was similar in diabetic and control animals when cholesterol was solubilized with 5 or 20 mM CDC or DC. These differences in cholesterol uptake using the various bile acids and the failure of CDC and DC to facilitate the enhanced uptake of cholesterol in diabetic animals remains unexplained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human gallbladder mucin binds biliary lipids and promotes cholesterol crystal nucleation in model bile

The binding of phosphatidylcholine and cholesterol in model bile to human gallbladder mucin was studied by means of a rapid filtration binding assay and sucrose density gradient ultracentrifugation. Numerous low affinity binding sites for phosphatidylcholine and cholesterol were present on gallbladder mucin. Binding of phosphatidylcholine and cholesterol to mucin increased as a function of cholesterol saturation index. Proteolytic digestion of mucin disaggregated the native mucin polymer and removed hydrophobic domains on the mucin peptide core that bind l-anilino-8-naphthalenesulfonic acid. Proteolytic digestion also resulted in a 91% and 78% decrease, respectively, in the binding of phosphatidylcholine and cholesterol to mucin. The ability of trypsin-treated and native mucin to promote the nucleation of cholesterol monohydrate crystals was compared in a model bile. The incidence of cholesterol monohydrate crystal nucleation with native mucin was significantly greater at 3 days than with trypsin-treated mucin or controls (P less than 0.001). After 3, 6, and 9 days of incubation, samples containing native mucin contained significantly more crystals than controls or trypsin-digested mucin samples (P less than 0.0001 for each). These data indicate that highly purified human gallbladder mucin binds phosphatidylcholine and cholesterol in model bile. Furthermore, this study demonstrates that structural integrity of the native mucin polymer and hydrophobic domains on the peptide core are essential for the nucleation of cholesterol monohydrate crystals by mucin in model bile.     

Cholesterol gallstones in alloxan-diabetic mice

Normal and alloxan-diabetic male mice (Crj-ICR) were fed a diet containing 0.5% cholesterol for 5 and 10 weeks, and gallbladder bile was analyzed for cholesterol, phospholipids and bile acids, feces for sterols and bile acids, and plasma and liver for cholesterol, phospholipids, and triglycerides. Normal mice developed no gallstones but the diabetic mice developed cholesterol gallstones with an incidence of 70% by 5 weeks and 80% by 10 weeks after feeding of the cholesterol diet. Diabetic mice fed the ordinary diet also developed stones (23%) by 10 weeks. In the diabetic mice, the gallbladder was enlarged about threefold, and biliary lipid concentration, diet intake, and fecal excretion of sterols and bile acids increased but body weight decreased. Cholic acid and beta-muricholic acid comprised over 40% each of the total biliary bile acids in normal mice, but cholic acid increased to about 80% and beta-muricholic acid decreased to a few percent in the diabetic mice. Fecal excretion of bile acids increased after cholesterol feeding in both normal and diabetic mice, but the increased bile acid in the normal animals was beta-muricholic acid and that in the diabetic mice was deoxycholic acid. The mice that developed gallstones showed a marked increase in biliary cholesterol value and decreases in gallbladder bile and bile acid concentration, but no difference in biliary and fecal bile acid composition, bile acid synthesis, fecal sterols, or plasma and liver lipid levels. Cholesterol absorption was increased in the diabetic mice when examined by plasma 14C/3H ratio and fecal 14C-labeled sterol excretion after a single oral administration of [14C]cholesterol and a simultaneous intravenous injection of [3H]cholesterol. These data led to the conclusion that cholesterol gallstones developed in alloxan-diabetic mice fed excess cholesterol, due to the hyperphagia and the enhancement of cholesterol absorption caused by increases in the synthesis and secretion of cholic acid.     

Bovine gallbladder mucin promotes cholesterol crystal nucleation from cholesterol-transporting vesicles in supersaturated model bile

This study examined the ability of purified gallbladder mucin to accelerate the nucleation of cholesterol monohydrate crystals from the cholesterol-transporting particles in supersaturated model bile. Mixed lipid micelles and cholesterol-phosphatidylcholine vesicles in supersaturated model bile were separated by Sephadex G-200 column chromatography. Mixed lipid micelles prepared by column chromatography had a low cholesterol-phosphatidylcholine ratio (0.30) and did not spontaneously nucleate cholesterol monohydrate crystals. In contrast, vesicles prepared by column chromatography had a cholesterol-phosphatidylcholine ratio of 1.00 and nucleated cholesterol crystals rapidly (P less than 0.001). Nucleation of cholesterol crystals was significantly accelerated in a concentration- and time-dependent manner by purified bovine gallbladder mucin in cholesterol containing vesicles, but not in mixed lipid micelles (P less than 0.001). A rapid filtration binding assay demonstrated significant binding of cholesterol and phosphatidylcholine in vesicles to gallbladder mucin but only minimal binding of cholesterol and phosphatidylcholine in mixed micelles. These data indicate that gallbladder mucin binds cholesterol and phosphatidylcholine in vesicles and accelerates the nucleation of cholesterol monohydrate crystals from these cholesterol-transporting particles in supersaturated model bile.     

Ileal bile acid transport regulates bile acid pool, synthesis, and plasma cholesterol levels differently in cholesterol-fed rats and rabbits

We investigated the effect of ileal bile acid transport on the regulation of classic and alternative bile acid synthesis in cholesterol-fed rats and rabbits. Bile acid pool sizes, fecal bile acid outputs (synthesis rates), and the activities of cholesterol 7alpha-hydroxylase (classic bile acid synthesis) and cholesterol 27-hydroxylase (alternative bile acid synthesis) were related to ileal bile acid transporter expression (ileal apical sodium-dependent bile acid transporter, ASBT). Plasma cholesterol levels rose 2.1-times in rats (98 +/- 19 mg/dl) and 31-times (986 +/- 188 mg/dl) in rabbits. The bile acid pool size remained constant (55 +/- 17 mg vs. 61 +/- 18 mg) in rats but doubled (254 +/- 46 to 533 +/- 53 mg) in rabbits. ASBT protein expression did not change in rats but rose 31% (P < 0.05) in rabbits. Fecal bile acid outputs that reflected bile acid synthesis increased 2- and 2.4-times (P < 0.05) in cholesterol-fed rats and rabbits, respectively. Cholesterol 7alpha-hydroxylase activity rose 33% (24 +/- 2.4 vs. 18 +/- 1.6 pmol/mg/min, P < 0.01) and mRNA levels increased 50% (P < 0.01) in rats but decreased 68% and 79%, respectively, in cholesterol-fed rabbits. Cholesterol 27-hydroxylase activity remained unchanged in rats but rose 62% (P < 0.05) in rabbits. Classic bile acid synthesis (cholesterol 7alpha-hydroxylase) was inhibited in rabbits because an enlarged bile acid pool developed from enhanced ileal bile acid transport. In contrast, in rats, cholesterol 7alpha-hydroxylase was stimulated but the bile acid pool did not enlarge because ASBT did not change. Therefore, although bile acid synthesis was increased via different pathways in rats and rabbits, enhanced ileal bile acid transport was critical for enlarging the bile acid pool size that exerted feedback regulation on cholesterol 7alpha-hydroxylase in rabbits.     

Vesicular cholesterol in bile. Relationship to protein concentration and nucleation time

A study was done to determine whether the nucleation time was related to the amount of cholesterol carried in vesicles. Bile was obtained from cholesterol gallstone patients and controls. Gel-exclusion chromatography was used to separate vesicles and micelles in the native bile using an eluting buffer containing 10 mM sodium cholate. The percent of total cholesterol carried in vesicles in gallbladder bile of stone patients was significantly greater than that in control patients. Total cholesterol concentration in gallbladder bile of stone patients was significantly greater than in controls. This difference was due to the fact that vesicular cholesterol concentration was significantly greater in the gallbladder bile of stone patients compared to controls. Micellar cholesterol concentrations were similar in the two groups. Nucleation time was related significantly to vesicular cholesterol concentration in correlation analysis and, as previously shown, so was total protein concentration. This study supports the importance of vesicular cholesterol in solid crystal formation and demonstrates for the first time that the rate of cholesterol monohydrate crystal formation is directly related to the amount of cholesterol transported in vesicles.     

Effect of streptozotocin-induced diabetes in neonatal rat on bile acid pool changes in adult life: selective sensitivity in females

The effect of streptozotocin-induced diabetes in neonatal rat on the bile acid pool and composition during adult life was investigated. Unlike the effect of diabetes in adult rats (where bile acid pool increases markedly), neonatal diabetes caused a reduction in bile acid pool in adult life in females (but not in males) with significant reduction in both cholic and chenodeoxycholic acids. Upon challenge with dietary cholesterol, only the female diabetic rat responded with a further reduction in total bile acid pool. These studies demonstrate a selective sensitivity in the female diabetic rat with regard to diabetes-induced changes in bile acid pool.     

Contraceptive steroids increase cholesterol in bile: mechanisms of action

Contraceptive steroids increase the risk of acquiring cholesterol gallstones. The factors responsible include an increase in cholesterol saturation of bile and an increase in rate of secretion of cholesterol into bile. The goal of this study was to investigate the mechanism(s) of these increases in biliary cholesterol. During the use of contraceptive steroids, cholesterol saturation of gallbladder bile and the amount of cholesterol secreted per mole of bile acid increased (P less than 0.05 and P less than 0.02, respectively). Cholesterol absorption, cholesterol synthesis, chylomicron remnant clearance, and the concentration of plasma and lipoprotein lipids were not altered by contraceptive steroids. Despite this apparent lack of effect, important correlations were present during steroid use. LDL (low density lipoprotein) cholesterol increased as dietary cholesterol increased (r = 0.58, P less than 0.025). Cholesterol synthesis correlated directly with VLDL cholesterol concentration (r = 0.64, P less than 0.01), biliary cholesterol secretion (r = 0.68, P less than 0.01) and with molar percent cholesterol in bile (r = 0.49, P = 0.06). Chylomicron remnant clearance also correlated with cholesterol secretion (r = 0.85, P less than 0.001). As either remnant uptake or synthesis increased, the effect of the other source of hepatic cholesterol on biliary cholesterol secretion diminished. These relationships were not observed in the same subjects when they were not taking the hormones. The findings suggest that both newly synthesized and dietary cholesterol contribute to the cholesterol secreted in bile. This is consistent with the hypothesis that cholesterol for secretion into bile and VLDL is derived from a common metabolic pool of free cholesterol. It is proposed that contraceptive steroids exert their effect on biliary cholesterol by increasing cholesterol entering the pool and/or by inhibiting hepatic ACAT (acylcoenzyme A:cholesterol acyltransferase) activity, a known effect of progesterone, so that an increase in free cholesterol entering the pool leads to an increase in output.     

Measurement of bile acid synthesis by 14CO2: the metabolism of propionyl CoA.

The relationship between 14CO2 evolution from the catabolism of [26 or 2714C] cholesterol to bile acids was studied in rats with biliary fistulae. When equal quantities of [26 or 2714C] cholesterol and [414C] cholesterol were administered, there was a significant linear relationship between 14CO2 expiration in the breath and [414C] bile acid excreted in the bile. Bile acid synthesis calculated as the ratio of 14CO2: molar specific activity of biliary cholesterol correlated highly with biliary bile acid excretion in the bile acid depleted rat. Phenobarbital, a known inducer of gamma-amino levulenic acid formation from succinyl CoA did not alter the relationship between the 14CO2 estimation of bile acid synthesis and biliary bile acid excretion, indicating that the relationship between [26 or 2714C] cholesterol side chain cleavage and 14CO2 formation was not altered. Phenobarbital, however, did cause a reduction in bile acid synthesis measured by 14CO2 evolution and by biliary bile acid excretion. The 14CO2 method underestimated bile acid excretion. 8.7% in untreated and phenobarbital treated rats respectively. Since 11% of the radioactivity which was expired as 14CO2 was isolated as bile acids, radioactivity cleaved as [1 or 314C] propionyl CoA may enter cholesterol-bile acid biosynthesis resulting in the underestimation of bile acid synthesis. To test whether radioactivity from propionyl CoA enters steroid biosynthesis [114C] propionate and [214C] propionate were given to untreated biliary fistula rats and the biliary lipids excreted in 60 hours were analyzed. Incorporation of radioactivity into cholesterol and bile acids was greater after the administration of [214C] propionate than after [114C] propionate than after [114C] propionate, suggesting that radioactivity from propionyl CoA may enter steroid biosynthesis by metabolic events in which the methylene and carboxyl carbon atoms are differentiated. Although the use of 14CO2 expiration from [26 or 2714C] cholesterol catabolism underestimates the rate of bile acid synthesis, it should have many applications because of the constant relationship between 14CO2 formation and cholesterol side chain cleavage.     

Cholecystectomy prevents expansion of the bile acid pool and inhibition of cholesterol 7alpha-hydroxylase in rabbits fed cholesterol

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

Thyroid hormone differentially augments biliary sterol secretion in the rat. II. The chronic bile fistula model.

To further define thyroid hormone effects on bile acid synthesis and biliary lipid secretion, studies were done in chronic bile fistula rats. Euthyroid and methimazole-hypothyroid rats, with and without triiodothyronine (T3) injection, had total bile diversion for timed bile collections. With interrupted enterohepatic circulation, cholesterol absorption is negligible and bile acid secretion equals bile acid synthesis rate. Hypothyroid rats had diminished levels of bile acid synthesis and biliary secretion of cholesterol and phospholipid. Single dose T3 injection produced a 13-fold increase in bile cholesterol secretion and a 3-fold increase in phospholipid secretion, both initiated 12 h after T3. Bile acid synthesis increased by 50%, but the increase did not begin until 24 h after T3. Neither hypothyroidism nor T3 treatment abolished diurnal rhythms of bile acid synthesis and biliary lipid secretion. Inhibition of cholesterol synthesis with lovastatin resulted in a persistent 33% decrease in bile acid synthesis in euthyroid and hypothyroid rats, while bile cholesterol secretion only transiently decreased. Inhibition of cholesterol synthesis did not alter T3-induced bile cholesterol secretion, with a 10-fold increase seen. However, bile acid synthesis was not stimulated by T3 in the presence of lovastatin. We conclude that facilitated bile acid synthesis and biliary cholesterol secretion are early effects of T3 and may account for the hypocholesterolemia of T3. Cholesterol synthesis does not appear to be required for the T3-induced bile cholesterol secretion.     

Examination of bile acid negative feedback regulation in rats

Recent data obtained using cultured rat hepatocytes showed that bile acids do not inhibit bile acid synthesis, whereas cholesterol concentrations vary in parallel with bile acid synthesis (Davis et al. (1983. J. Biol. Chem. 258: 4079-4082). This led us to re-evaluate in vivo experiments upon which the consensus that bile acid synthesis is primarily regulated by bile acid "negative feedback" is based. Infusion of taurocholate into either the jugular vein or duodenum of bile-diverted rats stimulated biliary cholesterol secretion and bile flow, but it did not inhibit bile acid synthesis. The lack of an inhibitory effect was evident using several different infusion rates of taurocholate. Even at the greatest rate of taurocholate infusion (25 mumol/(100 g.hr] there was no significant inhibition of bile acid synthesis. In contrast, infusing mevinolin (1 mg/hr), a potent competitive inhibitor of HMG-CoA reductase, almost completely inhibited bile acid synthesis and biliary cholesterol secretion. Since mevinolin did not affect bile flow, these results cannot be ascribed to bile secretory failure. Thus, while these studies suggest that taurocholate may not regulate bile acid synthesis directly via negative feedback, cholesterol is likely to act as a positive effector of bile acid synthesis.     

Development of bile acid metabolism: effect of glucocorticoids on bile acid metabolism in the neonatal rat

The objective of this study was to examine the effect of glucocorticoid treatment in early neonatal life on plasma cholesterol and hepatic cholesterol 7 alpha-hydroxylase (CH-7A), the rate-limiting enzyme of bile acid biosynthesis from cholesterol, measured at weaning (Postnatal Day 20). Neonatal rat pups were injected subcutaneously with 5 micrograms of dexamethasone (DEXA) or vehicle (CON) for 5 days between Postnatal Days 4 and 8. On Postnatal Day 20, the animals were used for various studies. DEXA-treated pups weighed significantly less (P less than 0.001) than controls. Even though DEXA-treated animals had significantly smaller livers (P less than 0.001), microsomal protein per gram of liver was significantly greater (P less than 0.005) in the DEXA-treated animals. CH-7A activity (pmole/mg . min) was significantly lower (P less than 0.005) in the DEXA-treated animals (CON (4) 19.4 +/- 2.8; DEXA (4) 5.0 +/- 1.0). Plasma cholesterol (mg/100 ml) was significantly greater (P less than 0.005) in the DEXA-treated animals (CON (5) 179 +/- 7; DEXA (4) 223 +/- 5), a finding consistent with lower CH-7A activity in this group. Taurocholate absorption by in situ ileal loops in anesthetized rats was significantly greater in the DEXA-treated animals in agreement with the in vitro observations of Little and Lester. The basis for the reduced CH-7A activity in DEXA-treated pups is not known. It may be due in part to a new steady state in the enterohepatic circulation of bile acids resulting from a glucocorticoid-induced enhanced conservation of bile acids.     

Regulation of bile acid synthesis. III. Correlation between biliary bile salt hydrophobicity index and the activities of enzymes regulating cholesterol and bile acid synthesis in the rat

Hepatic bile acid synthesis is thought to be under negative feedback control by bile salts in the enterohepatic circulation, acting at the level of cholesterol 7 alpha-hydroxylase (C7 alpha H), the initial and rate-limiting step in the bile acid biosynthetic pathway. Bile salts also suppress the activity of the rate-limiting enzyme for cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA-R). The mechanisms of these regulatory effects are poorly understood, and one or both may be indirect. Previous data suggest that the hydrophilic-hydrophobic balance of bile salts, a major determinant of their cholesterol solubilizing properties, also determines their potency as regulators of bile acid and cholesterol synthesis. To further evaluate the relationship between the physicochemical and regulatory properties of bile acids, we altered the composition of the bile salt pool of rats by feeding one or more of seven different bile acids (1% w/w for 14 days). We then determined the mean hydrophilic-hydrophobic balance (hydrophobicity index) of the bile salts in bile, and correlated this with the specific activities of C7 alpha H and HMG-CoA-R, and of acyl-CoA:cholesterol acyltransferase (ACAT), a third hepatic microsomal enzyme which regulates cholesterol esterification. In all instances following bile acid feeding, conjugates of the fed bile acid(s) became the predominant bile salts in bile. Highly significant negative linear correlations (each P less than 0.0001) were found between the hydrophobicity indices of biliary bile salts and the activities of C7 alpha H (r = 0.79) or HMG-CoA-R (r = 0.63). By contrast, no significant correlation could be demonstrated between ACAT activity and the hydrophobicity index of biliary bile salts. The correlation between activities of HMG-CoA-R and C7 alpha H was also highly significant (r = 0.81; P less than 0.0001). No significant correlation existed between ACAT and either HMG-CoA-R or C7 alpha H. Microsomal free cholesterol was not consistently altered by bile acid feeding. Thus, the potency of circulating bile salts as suppressors of the enzymes regulating bile acid and cholesterol synthesis increases with increasing hydrophobicity. The hydrophobic-hydrophilic balance of the bile salt pool may play an important role in the regulation of cholesterol and bile acid synthesis.     

Absence of negative feedback control of bile acid biosynthesis in cultured rat hepatocytes

The effect of individual bile acids on bile acid synthesis was studied in primary hepatocyte cultures. Relative rates of bile acid synthesis were measured as the conversion of lipoprotein [4-14C]cholesterol into 4-14C-labeled bile acids. Additions to the culture media of cholate, taurocholate, glycocholate, chenodeoxycholate, taurochenodeoxycholate, glycochenodeoxycholate, deoxycholate, and taurodeoxycholate (10-200 microM) did not inhibit bile acid synthesis. The addition of cholate (100 microM) to the medium raised the intracellular level of cholate 10-fold, documenting effective uptake of added bile acid by cultured hepatocytes. The addition of 200 microM taurocholate to cultured hepatocytes prelabeled with [4-14C]cholesterol did not result in inhibition of bile acid synthesis. Taurocholate (10-200 microM) also failed to inhibit bile acid synthesis in suspensions of freshly isolated hepatocytes after 2, 4, and 6 h of incubation. Surprisingly, the addition of taurocholate and taurochenodeoxycholate (10-200 microM) stimulated taurocholate synthesis from [2-14C]mevalonate-labeled cholesterol (p less than 0.05). Neither taurocholate nor taurochenodeoxycholate directly inhibited cholesterol 7 alpha-hydroxylase activity in the microsomes prepared from cholestyramine-fed rats. By contrast, 7-ketocholesterol and 20 alpha-hydroxycholesterol strongly inhibited cholesterol 7 alpha-hydroxylase activity at low concentrations (10 microM). In conclusion, these data strongly suggest that bile acids, at the level of the hepatocyte, do not directly inhibit bile acid synthesis from exogenous or endogenous cholesterol even at concentrations 3-6-fold higher than those found in rat portal blood.     

Stability of mixed micellar bile models supersaturated with cholesterol.

The maximal equilibrium solubility of cholesterol in mixtures of phosphatidylcholine (PC)1 and bile salts depends on the cholesterol/PC ratio (Rc) and on the effective ratio (Re) between nonmonomeric bile salts and the sum (CT) of PC and cholesterol concentrations (Carey and Small, 1978; Lichtenberg et al., 1984). By contrast, the concentration of bile salts required for solubilization of liposomes made of PC and cholesterol does not depend on Rc (Lichtenberg et al., 1984 and 1988). Thus, for Rc greater than 0.4, solubilization of the PC-cholesterol liposomes yields PC-cholesterol-bile salts mixed micellar systems which are supersaturated with cholesterol. In these metastable systems, the mixed micelles spontaneously undergo partial revesiculation followed by crystallization of cholesterol. The rate of the latter processes depends upon Rc, Re, and CT. For any given Rc and Re, the rate of revesiculation increases dramatically with increasing the lipid concentration CT, reflecting the involvement of many mixed micelles in the formation of each vesicle. The rate also increases, for any given CT and Re, upon increasing the cholesterol to PC ratio, Rc, probably due to the increasing degree of supersaturation. Increasing the cholate to lipid effective ratio, Re, by elevation of cholate concentration at constant Rc and CT has a complex effect on the rate of the revesiculation process. As expected, cholate concentration higher than that required for complete solubilization at equilibrium yields stable mixed micellar systems which do not undergo revesiculation, but for lower cholate concentrations decreasing the degree of supersaturation (by increasing [cholate]) results in faster revesiculation. We interpret these results in terms of the structure of the mixed micelles; micelles with two or more PC molecules per one molecule of cholesterol are relatively stable but increasing the bile salt concentration may cause dissociation of such 1:2 cholesterol:PC complexes, hence reducing the stability of the mixed micellar dispersions. The instability of PC-cholesterol-cholate mixed systems with intermediary range of cholate to lipids ratio may be significant to gallbladder stone formation as: (a) biliary bile contains PC-cholesterol vesicles which may be, at least partially, solubilized by bile salts during the process of bile concentration in the gallbladder, resulting in mixtures similar to our model systems; and (b) the bile composition of cholesterol gallstone patients is within an intermediary range of bile salts to lipids ratio.     

Cholesterol and bile acid dynamics: comparative aspects

While the cholesterol concentration in a given tissue is similar in the rat, pig or man, the relative importance of the processes regulating the input (absorption and synthesis) and output (faecal cholesterol and bile acid excretions) of the cholesterol system is very different from one species to another. The rat, whose cholesterolaemia does not significantly increase after cholesterol addition to the diet ("hyporesponding" animal), successfully adapts its bile acid biosynthesis to variations in cholesterol input. This process accounts for 80 to 85% of cholesterol output, faecal cholesterol excretion being a minor process. The latter results from a low liver cholesterol secretion in the bile due to the low hydrophobicity of its main bile acids. Furthermore, in this animal a high intestinal synthesis of cholesterol and apolipoproteins (particularly B48) is observed. The latter are secreted as very light lipoproteins (chylomicrons and VLDL) with a faster plasma turnover than the VLDL (apoB100, E...) secreted by the liver. The "remnants" of rat VLDL are essentially very rapidly taken up by the liver; their interplasmatic transformation pathway into IDL and LDL is not very significant (less than or equal to 10%). Man, who has a more significant hypercholesterolaemia after exogenous cholesterol ingestion ("hyperresponding" subject) seems to have a less modulable capacity for transforming cholesterol into bile acids. This process accounts for only 50% of cholesterol output, faecal cholesterol excretion being quantitatively just as significant. Cholesterol concentration and the cholesterol/bile acid ratio are much higher in human than in rat bile, the main bile acids being more hydrophobic. While both the intestine and liver contribute to cholesterogenesis, the relative importance of the latter is probably greater in man than in the rat. Moreover, a larger fraction of plasma VLDL is transformed into IDL and LDL, the latter representing the main plasma cholesterol carrier. Determining whether the differences between the biodynamics of cholesterol processes in the rat and in man can be generalised to mammals with low or high sensitivities to hypercholesterolaemia and atherosclerosis seems to be a fundamental research objective for the next few years.     

Effect of bile duct ligation on bile acid and cholesterol metabolism in rats

The effects of bile duct ligation on bile acid and cholesterol metabolism were examined in male Wistar strain rats. Quantitative and qualitative changes of bile acids and cholesterol in serum and urine occurred; beta-muricholic acid predominantly increased in serum and urine and the ratio of urinary cholic acid and beta-muricholic acid changed from about 5:3 on day 1 to about 1:8 on day 5 under biliary obstruction. The form of the increased urinary bile acids was mainly taurine-conjugated and partly sulfated. Under conditions of bile duct ligation on day 5, 14C-labeled 3 beta-hydroxy-5-cholenoic, lithocholic, and chenodeoxycholic acids were intragastrically administered to the rats after pretreatment with antibiotics and the metabolites of these three acids were investigated. 3 beta-Hydroxy-5-cholenoic acid was most efficiently converted to beta-muricholic acid. The present study strongly suggested the presence of an alternative metabolic pathway induced by bile duct ligation, which caused the change in composition of urinary bile acids, and especially the marked increase in beta-muricholic acid formation. A possible alternative pathway for bile acid biosynthesis under biliary obstruction in rats is postulated.     

Megalin and cubilin expression in gallbladder epithelium and regulation by bile acids

Cholesterol crystal formation in the gallbladder is a key step in gallstone pathogenesis. Gallbladder epithelial cells might prevent luminal gallstone formation through a poorly understood cholesterol absorption process. Genetic studies in mice have highlighted potential gallstone susceptibility alleles, Lith genes, which include the gene for megalin. Megalin, in conjunction with the large peripheral membrane protein cubilin, mediates the endocytosis of numerous ligands, including HDL/apolipoprotein A-I (apoA-I). Although the bile contains apoA-I and several cholesterol-binding megalin ligands, the expression of megalin and cubilin in the gallbladder has not been investigated. Here, we show that both proteins are expressed by human and mouse gallbladder epithelia. In vitro studies using a megalin-expressing cell line showed that lithocholic acid strongly inhibits and cholic and chenodeoxycholic acids increase megalin expression. The effects of bile acids (BAs) were also demonstrated in vivo, analyzing gallbladder levels of megalin and cubilin from mice fed with different BAs. The BA effects could be mediated by the farnesoid X receptor, expressed in the gallbladder. Megalin protein was also strongly increased after feeding a lithogenic diet. These results indicate a physiological role for megalin and cubilin in the gallbladder and provide support for a role for megalin in gallstone pathogenesis.     

Effect of dietary fenugreek seeds on biliary proteins that influence nucleation of cholesterol crystals in bile

Formation of cholesterol gallstones in gallbladder is controlled by procrystallising and anticrystallising factors present in bile. Dietary fenugreek seed has been recently observed to possess anti-lithogenic potential in experimental mice. In the current animal study, we evaluated the effect of dietary fenugreek on the compositional changes in the bile, particularly its effect on glycoproteins, low-molecular-weight (LMW) and high-molecular-weight (HMW) proteins, cholesterol nucleation time and cholesterol crystal growth. Groups of Wistar rats were fed for 10 weeks with diets: (1) basal control (C), (2) C + fenugreek (12%), (3) high cholesterol diet (HCD) and (4) HCD + fenugreek (12%). Feeding of HCD containing 0.5% cholesterol for 10 weeks rendered the bile lithogenic. Incorporation of fenugreek into HCD decreased the cholesterol content (70.5%), total protein (58.3%), glycoprotein (27.5%), lipid peroxides (13.6%) and cholesterol saturation index (from 1.98 to 0.75) in bile, increased the bile flow rate (19.5%), prolonged the cholesterol nucleation time and reduced the vesicular form of cholesterol (65%), which was accompanied with an increase in smaller vesicular form (94%). There was an increase in biliary phospholipid (33%) and total bile acid (49%) contents in the HCD + fenugreek group as compared with the HCD group. Electrophoretic separation of biliary LMW proteins showed the presence of a high concentration of 28-kDa protein, which might be responsible for the prolongation of cholesterol nucleation time in the fenugreek-fed groups. These findings indicate that the beneficial anti-lithogenic effect of dietary fenugreek, which primarily is due to reduction in the cholesterol content in bile, was additionally affected through a modulation of the nucleating and anti-nucleating proteins, which, in turn, affect cholesterol crystallisation.