Regulation of rat biliary cholesterol secretion by agents that alter intrahepatic cholesterol metabolism. Evidence for a distinct biliary precursor pool.

Propensity for cholesterol gallstone formation is determined in part by biliary cholesterol content relative to bile salts and phospholipid. We examined the hypothesis that the rate of biliary cholesterol secretion can be controlled by availability of an hepatic metabolically active free cholesterol pool whose size is determined in part by rates of sterol synthesis, as reflected by activity of the primary rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and of sterol esterification, as reflected by the activity of the enzyme acyl coenzyme A/cholesterol acyltransferase (ACAT). Rats were prepared with biliary, venous, and duodenal catheters. The enterohepatic circulation of biliary lipids was maintained constant by infusion of a bile salt, lecithin, cholesterol replacement solution. Administration of 25-hydroxycholesterol decreased HMG CoA reductase activity, increased ACAT activity, and decreased biliary cholesterol output 26% by 1 h. By 2 h, ACAT activity and biliary cholesterol secretion were at control levels. Administration of mevinolin, a competitive inhibitor of HMG CoA reductase, had no effect on ACAT activity and decreased biliary cholesterol secretion 16%. Administration of progesterone, an inhibitor of ACAT, had no effect on HMG CoA reductase and increased biliary cholesterol output 32% at 1 h. By 2 h, all parameters were near control levels. None of these agents had any significant effect on biliary bile salt or phospholipid secretion. Thus, acutely altering rates of esterification and/or synthesis can have profound effects on biliary cholesterol secretion independent of the other biliary lipids. These experiments suggest the existence of a metabolically active pool of free cholesterol that serves as a precursor pool for biliary cholesterol secretion. Furthermore, the size of this precursor pool is determined in part both by rates of cholesterol synthesis and esterification and is a key determinant of biliary cholesterol secretion.     

Mechanisms of gallstone formation in women. Effects of exogenous estrogen (Premarin) and dietary cholesterol on hepatic lipid metabolism.

Our aim was to define mechanisms whereby conjugated estrogens (Premarin, exogenous estrogen; Ayerst Laboratories, New York) increase the risk of developing cholesterol gallstones and to determine the role, if any, of dietary cholesterol. We studied gallbladder motor function, biliary lipid composition and secretion, cholesterol absorption, cholesterol synthesis and esterification by peripheral blood mononuclear cells, the clearance of chylomicron remnants, and bile acid kinetics in 29 anovulatory women. 13 were studied on both a low (443 +/- 119 mumol/d) and high (2,021 +/- 262 mumol/d) cholesterol diet. Premarin increased the lithogenic index of bile (P less than 0.05), increased biliary cholesterol secretion (P less than 0.005), lowered chenodeoxycholate (CDCA) pool (P less than 0.001) and synthesis (P less than 0.05), altered biliary bile acid composition [( CA + DCA]/CDCA increases, P less than 0.005), stimulated cholesterol esterification (P less than 0.03), and enhanced the clearance of chylomicron remnants (P = 0.07). Increases in dietary cholesterol stimulated the biliary secretion of cholesterol (P = 0.07), bile acid (P less than 0.05), phospholipid (P = 0.07), and as a result, did not alter lithogenic index. The reduction in CDCA pool and synthesis by Premarin was reversed by increasing dietary cholesterol. Off Premarin, only 24% of the increase in cholesterol entering the body in the diet was recovered as biliary cholesterol or newly synthesized bile acid. On Premarin, 68% of this increase in cholesterol was recovered as these biliary lipids. We conclude that Premarin increases biliary cholesterol by enhancing hepatic lipoprotein uptake and inhibiting bile acid synthesis. These actions of Premarin divert dietary cholesterol into bile.     

Regulation of biliary cholesterol secretion. Functional relationship between the canalicular and sinusoidal cholesterol secretory pathways in the rat.

The functional interrelationship between biliary cholesterol secretion, sinusoidal lipoprotein cholesterol secretion and bile salt synthesis was studied in the rat. Diosgenin, fructose, and colestipol in the diet were used to, respectively, influence biliary cholesterol output, VLDL production and bile salt synthesis. In the acute bile fistula rat, biliary cholesterol output was 700% increased by diosgenin and 50% decreased by fructose. In the rats fed both diosgenin and fructose, biliary cholesterol secretion was increased only by approximately 200%, whereas biliary bile salts and phospholipid outputs were unchanged. In the isolated perfused liver, VLDL-cholesterol output was 50% reduced by diosgenin alone, but was unchanged following feeding of diosgenin plus fructose. However, the livers of rats fed diosgenin plus fructose exhibited a 700% increase in VLDL-triglyceride production and a 200% increase in VLDL-cholesterol output. A significant reciprocal relationship between VLDL-cholesterol secretion and the coupling ratio of cholesterol to bile salts in bile was observed. Colestipol added to the diet maintained both sinusoidal and biliary cholesterol outputs within the normal range. In the chronic bile fistula rat, colestipol increased bile salt synthesis by 100% while diosgenin and fructose diets had no effect. Similarly, the addition of fructose to the colestipol diet did not decrease bile salt synthesis. These data suggest a reciprocal relationship between biliary cholesterol secretion and hepatic secretion of cholesterol as VLDL particles. The free cholesterol pool used for bile salt synthesis seems functionally unrelated to the pool from which VLDL-cholesterol and biliary cholesterol originate. These findings support the idea that metabolic compartmentalization of hepatic cholesterol is a major determinant of the quantity of cholesterol available for recruitment by the bile salt-dependent biliary cholesterol secretory mechanism.     

Biliary lipid excretion in patients with pigment gallstones. A comparison with cholesterol gallstone patients

Pigment gallstone patients are believed to have normal biliary lipid excretion. In order to measure this and to better understand cholesterol gallstone formation, the kinetics of biliary lipid excretion were studied in three patients who had been cholecystectomized for pigment gallstones and the results compared to those previously obtained in patients cholecystectomized for cholesterol gallstone. Pigment-stone patients had hyperbolic relationships between cholesterol and phospholipid outputs and bile salt output which were similar to those seen in cholesterol-stone patients. However, pigment-stone patients excreted more cholesterol and phospholipid at high bile salt output but approached those levels more gradually than cholesterol-stone patients. As a result, pigment-stone patients produced bile undersaturated with cholesterol at a lower bile salt output than cholesterol-stone patients, and thus they would be less likely to produce supersaturated bile during low bile salt output such as that occurring during an overnight fast. The data suggest that cholesterol-stone patients, in addition to excreting more cholesterol and less bile salts than normals, have a defect in the rate of lipid output in response to decreasing bile salt output.     

Biliary copper excretion by hepatocyte lysosomes in the rat. Major excretory pathway in experimental copper overload.

We investigated the hypothesis that lysosomes are the main source of biliary copper in conditions of hepatic copper overload. We used a rat model of oral copper loading and studied the relationship between the biliary output of copper and lysosomal hydrolases. Male Sprague-Dawley rats were given tap water with or without 0.125% copper acetate for up to 36 wk. Copper loading produced a 23-fold increase in the hepatic copper concentration and a 30-65% increase in hepatic lysosomal enzyme activity. Acid phosphatase histochemistry showed that copper-loaded livers contained an increased number of hepatocyte lysosomes; increased copper concentration of these organelles was confirmed directly by both x ray microanalysis and tissue fractionation. The copper-loaded rats showed a 16-fold increase in biliary copper output and a 50-300% increase in biliary lysosomal enzyme output. In the basal state, excretory profiles over time were similar for biliary outputs of lysosomal enzymes and copper in the copper-loaded animals but not in controls. After pharmacologic stimulation of lysosomal exocytosis, biliary outputs of copper and lysosomal hydrolases in the copper-loaded animals remained coupled: injection of colchicine or vinblastine produced an acute rise in the biliary output of both lysosomal enzymes and copper to 150-250% of baseline rates. After these same drugs, control animals showed only the expected increase in lysosomal enzyme output without a corresponding increase in copper output. We conclude that the hepatocyte responds to an increased copper load by sequestering excess copper in an increased number of lysosomes that then empty their contents directly into bile. The results provide direct evidence that exocytosis of lysosomal contents into biliary canaliculi is the major mechanism for biliary copper excretion in hepatic copper overload.     

Effects of Bay o 2752, a hypocholesterolemic agent, on intestinal taurocholate absorption and cholesterol esterification

Bay o 2752 [N,N'-(1, 11-undecandiyl)bis(2,3-dihydro-2-methyl-1H-indole-1-carboxamide)] has been demonstrated in rats to inhibit intestinal cholesterol absorption. Studies were carried out in male Wistar rats to determine if the mechanism is inhibition of intestinal bile acid absorption or cholesterol esterification. Bay o 2752 did not alter intestinal bile acid absorption as measured by in vitro uptake of [14C]taurocholic acid into ileal everted sacs (0.01 and 1.0 mg/ml of Bay o 2752) or the biliary excretion of radioactivity after in vivo ileal perfusion of the bile acid and drug (1.0 mg/ml at 1.0 ml/min for 1 hr). Cholesterol esterification was determined by measurement of in vitro activity of acyl coenzyme A:cholesterol acyltransferase from hepatic microsomes and cholesterol ester hydrolase from pancreatic supernatant, and the in vivo lymphatic output of cholesteryl ester after intraduodenal cholesteryl infusion. Addition of Bay o 2752 (0.01-10 micrograms/ml) to hepatic microsomes produced a concentration-dependent decrease in acyl coenzyme A:cholesterol acyltransferase activity with an IC50 of 0.95 micrograms/ml. Cholesterol ester hydrolase activity was unaffected by the drug (1.0-100 micrograms/ml). Intraduodenal infusion of Bay o 2752 (10 mg/ml at 0.9 ml/hr for 8 hr) reduced markedly the flux of cholesterol from the intestinal lumen into the mesenteric lymph, especially the lymphatic output of the esterified form of both radioisotopically labeled and total cholesterol. These data suggest that Bay o 2752-induced reduction in intestinal cholesterol absorption results from its potent inhibitory effect on acyl coenzyme A:cholesterol acyltransferase activity.     

The formation of abnormal bile and cholesterol gallstones from dietary cholesterol in the prairie dog

To study the pathogenesis of cholesterol gallstones, we fed 24 adult male prairie dogs a high cholesterol, egg yolk diet. 13 control animals received a cholesterol-free diet. All animals fed the egg yolk diet formed multiple gallstones in 2-6 months' time. These stones contained cholesterol, 77+/-14% by dry weight. No stones ocurred in the control group.The egg yolk-fed animals developed bile of altered chemical composition. The cholesterol concentration of hepatic and gallbladder bile increased significantly. The molar ratios of bile acid/cholesterol and phospholipid/cholesterol decreased in hepatic and gallbladder bile. The predominant bile acid shifted from cholic acid, 78% of the total bile acids, to chenodeoxycholic acid, 60% of the total. In common bile duct cannulated animals the high cholesterol diet produced increased secretion of cholesterol by the liver and increased bile flow.In animals fed the egg yolk diet for 2 months, cholesterol-4-(14)C was included in the daily diet for the next 4 months to establish an isotopic steady state. At autopsy the mean specific activity of cholesterol was similar in serum, liver, hepatic bile, gallbladder bile, and gallstones. Thus the cholesterol of gallstones apparently equilibrated constantly throughout the study and was not sequestrated as a static pool.The high cholesterol, egg yolk diet caused the secretion of an "abnormal bile" which led to precipitation of cholesterol from micellar solution. The increased bile cholesterol relative to bile acid and phospholipid favored stone formation. This dietary induction of cholesterol gallstones provided a unique animal model, in part but not completely analogous to human cholelithiasis.     

Protoporphyrin IX-induced impairment of biliary lipid secretion in the rat

1. In order to gain information on the effect of protoporphyrin IX on changes in the properties of the canalicular plasma membrane, we studied the release of canalicular membrane constituents, namely phospholipids, cholesterol and 5'-nucleotidase, into bile in anaesthetized rats receiving saline or taurocholate (0.5 mumol min-1 100 g-1 body weight) with or without protoporphyrin IX infusion (10 or 20 micrograms min-1 100 g-1 body weight). 2. Protoporphyrin IX induced an impairment of spontaneous bile flow and of biliary secretion of cholesterol, phospholipids and bile acids. The taurocholate-induced increase in bile acid output was not significantly reduced by protoporphyrin IX at either of the doses used. However, when a cholestatic dose of protoporphyrin IX was infused, the taurocholate-induced bile flow and secretion of lecithin and cholesterol were significantly reduced. 3. Biliary output of phospholipid species other than lecithin did not counterbalance the protoporphyrin IX-induced reduction in biliary lecithin secretion. Biliary outputs of both total phospholipid and lecithin were inhibited by protoporphyrin IX to similar extents. 4. Protoporphyrin IX alone had no effect on the biliary release of 5'-nucleotidase, whereas when it was given with taurocholate, it increased the bile acid-induced biliary output of this enzyme markedly. 5. In summary, these results indicate that protoporphyrin IX impairs the biliary secretion of phospholipids and cholesterol but not that of bile acid. The release of canalicular membrane constituents other than lipids was also modified by protoporphyrin IX.     

Kinetic analysis of biliary lipid excretion in man and dog.

To understand better the mechanisms involved in biliary lipid excretion and to evaluate their role in cholesterol gallstone formation, the rates of biliary excretion of bile salts, cholesterol, and phospholipids were measured in two species, man and dog. Seven cholecystectomized patients with balloon-occludable reinfusion T-tubes were studied during intact and interrupted enterohepatic circulation and four cholecystectomized dogs were studied during interrupted enterohepatic circulation. In man and dog both cholesterol and phospholipid outputs were hyperbolically related to bile salt output by the equation y = x/(a + bx). The output curves intersected the origin and showed an initial rapid rise, followed by a slower increase to a maximum, suggesting a rate-limited mechanism. The shape of the curves permitted calculation of the theoretical maximal outputs and the rates of rise to those outputs. Comparison of these values showed that in both man and dog phospholipid output was greater than cholesterol output and that cholesterol and phospholipid were excreted at different rates. These studies (a) indicate that cholesterol, phospholipids, and bile salts are not excreted in a fixed relationship and (b) demonstrate the usefulness of the derived theoretical maximal lipid output, and the rate of rise of lipid excretion to a maximum, in evaluating the kinetics of biliary lipid excretion.     

Cholestasis induced by sulphated glycolithocholic acid in the rat: protection by endogenous bile acids

Sulphated glycolithocholic acid (SGLC) causes cholestasis in experimental animals, despite its sulphated form. In the present study, the cholestatic potency and the pharmacokinetics of SGLC were investigated in rats under two conditions: (a) in the presence of an intact circulating bile acid pool and (b) after exhaustion of the bile acid pool by 24 h of bile diversion. Intravenous administration of SGLC (8 mumol/100 g body weight) to rats with an intact bile acid pool did not cause cholestasis. However, biliary phospholipid and cholesterol concentrations were reduced by 40% and 29% respectively during the first hour after administration. When the same dose of the bile acid was injected in rats with a 24 h biliary drainage, a complete cessation of bile production was observed within 1 h. Twelve hours after the onset of cholestasis, bile production gradually increased again, showed a marked overshoot, and reached control levels after 3 days. In the recovery phase, biliary phospholipid and cholesterol concentrations were greatly reduced. The absence of endogenous bile acids did not change the hepatic clearance rate of a tracer dose of radiolabelled SGLC, but markedly decreased its biliary excretion rate. It was concluded that the hepatotoxic effect of SGLC is much more pronounced in rats with an exhausted bile acid pool, possibly due to a slower biliary excretion of the toxic compound. This phenomenon may have clinical implications for patients with a contracted bile acid pool.     

The mechanisms of and the interrelationship between bile acid and chylomicron-mediated regulation of hepatic cholesterol synthesis in the liver of the rat.

Hepatic cholesterol synthesis is controlled by both the size of the bile acid pool in the enterohepatic circulation and by the amount of cholesterol reaching the liver carried in chylomicron remnants. These studies were undertaken to examine how these two control mechanisms are interrelated. When the size of the pool was systematically varied, the logarithm of the rate of hepatic cholesterol synthesis varied in an inverse linear fashion with the size of the taurocholate pool between the limits of 0 and 60 mg of bile acid per 100 g of body weight. The slope of this relationship gave the fractional inhibition of cholesterol synthesis associated with expansion of the taurocholate pool and was critically dependent upon the amount of cholesterol available for absorption from the gastrointestinal tract. Furthermore, the degree of inhibition of cholesterol synthesis in the liver seen with taurocholate feeding was reduced by partially blocking cholesterol absorption with beta-sitosterol even though the bile acid pool was still markedly expanded. In rats with diversion of the intestinal lymph from the blood, a five-fold expansion of the taurocholate pool resulted in only slight suppression of the rate of hepatic cholesterol synthesis, and even this inhibition was shown to be attributable to small amounts of cholesterol absorbed through collateral lymphatic vessels and (or) to a fasting effect. Similarly, the infusion of either taurocholate or a combination of taurocholate and taurochenate into rats with no biliary or dietary cholesterol available for absorption caused no suppression of hepatic cholesterol synthesis. Finally, the effect of changes in the rate of bile acid snythesis on hepatic cholesterol synthesis was examined. The fractional inhibition of cholesterol synthesis found after administration of an amount of cholesterol sufficient to raise the hepatic cholesterol ester content by 1 mg/g equalled only --0.36 when bile acid snythesis was increased by biliary diversion but was --0.92 when bile acid synthesis was suppressed by bile acid feeding. It is concluded that (a) bile acids are not direct effectors of the rate of hepatic cholesterol synthesis, (b) most of the inhibitory activity seen with bile acid feeding is mediated through increased cholesterol absorption, and (c) bile acids do have an intrahepatic effect in that they regulate hepatic cholesterol synthesis indirectly by altering the flow of cellular cholesterol to bile acids.     

Effects of obesity and caloric intake on biliary lipid metabolism in man.

The effects of obesity and caloric intake on biliary lipid metabolism were investigated in a series of related studies. The degree of saturation of gallbladder bile with cholesterol was found to be significantly higher in a group of 23 obese healthy subjects than in a group of 23 nonobese controls matched for age, sex, and race. Bile was also significantly more saturated in 11 obese subjects before than after weight reduction. To determine whether supersaturated bile in obesity is due to excessive secretion of cholesterol or to deficient secretion of bile acids and phospholipids, the hepatic outputs of these three lipids were measured during constant duodenal infusion of formula in the same 11 subjects before and after weight reduction. Weight reduction resulted in significant reduction of cholesterol output but not of bile acid or phospholipid output. Moreover, very obese subjects were found to have cholesterol secretion rates markedly higher than less obese subjects previously studied by the same method. In obese subjects, bile was supersaturated with cholesterol despite increased bile acid pool sizes and increased secretion rates of bile acids and phospholipids. Supersaturated bile in the obese could therefore be attributed to a single defect in lipid secretion, namely, an excessive output of cholesterol. To determine whether the rate of caloric intake can account for the effects of obesity on biliary lipid composition and secretion, nine obese white men were studied on a weight maintenance diet and then during weight reduction on a 1,000 cal diet. As compared to weight maintenance, chronic caloric restriction resulted in reduced outputs of cholesterol, bile acids, and phospholipids, reduced bile acid pool size, and reduced synthesis and fecal excretion of cholesterol. Saturation of bile with cholesterol did not decrease during weight reduction, evidently because of the mobilization of cholesterol from adipose stores and the marked reduction in bile acid and phospholipid output observed during chronic caloric restriction. Acute alterations in caloric infusion rates did not fully reproduce the effects of chronic administration of high and low calorie diets. Likewise, chronic intake of hypercaloric diets by nonobese subjects did not reproduce the cholesterol hypersecretion characteristic of the obese. Thus, increased cholesterol secretion in obese subjects could not be fully explained by the amount of calories they ingested to maintain stable weight. It is concluded that obesity is characterized by excessive hepatic secretion of cholesterol which results in supersaturated bile.     

Separate transport systems for biliary secretion of sulfated and unsulfated bile acids in the rat.

Biliary secretion of 3 alpha-sulfated bile acids has been studied in Wistar rats with an autosomal recessive defect in the hepatic transport of bilirubin. Liver function, established by measurement of various enzymes in plasma, by enzyme histochemical methods, and by electron microscopy, appeared to be normal in these rats. Serum levels of unconjugated, monoglucuronidated, and diglucuronidated bilirubin were 0.62, 1.62, and 6.16 mumol/liter, respectively, compared with 0.17, 0.08, and 0.02 mumol/liter in control rats. Biliary bilirubin secretion was strongly reduced in the mutant animals: 0.21 +/- 0.03 vs. 0.39 +/- 0.03 nmol/min per 100 g body wt in control rats. Despite normal biliary bile acid output, bile flow was markedly impaired in the mutant animals, due to a 53% reduction of the bile acid-independent fraction of bile flow. The transport maximum for biliary secretion of dibromosulphthalein (DBSP) was also drastically reduced (-53%). Biliary secretion of intravenously administered trace amounts of the 3 alpha-sulfate esters of 14C-labeled taurocholic acid (-14%), taurochenodeoxycholic acid (-39%), taurolithocholic acid (-73%), and glycolithocholic acid (-91%) was impaired in the jaundiced rats compared with controls, in contrast to the biliary secretion of the unsulfated parent compounds. Hepatic uptake of sulfated glycolithocholic acid was not affected in the jaundiced animals. Preadministration of DBSP (15 mumol/100 g body wt) to normal Wistar rats significantly impaired the biliary secretion of sulfated glycolithocholic acid, but did not affect taurocholic acid secretion. We conclude that separate transport systems in the rat liver exist for biliary secretion of sulfated and unsulfated bile acids; the sulfates probably share secretory pathways with the organic anions bilirubin and DBSP. The described genetic defect in hepatic transport function is associated with a reduced capacity to secrete sulfated bile acids into bile; this becomes more pronounced with a decreasing number of hydroxyl groups on the sulfated bile acid's molecule.     

Dietary Factors Affecting Biliary Lipid Secretion in the Rhesus Monkey A Mechanism for the Hypocholesterolaemic Action of Polyunsaturated Fat?*

Abstract Using an experimental model which permits representative bile sampling whilst maintaining an almost “intact” enterohepatic circulation, it was found that different diets markedly influenced biliary lipid secretion in the rhesus monkey. In different studies, we have previously shown that bile volume, bile salt, phospholipid and cholesterol secretion rates were significantly less in animals given a low fat commercial diet then in monkeys given a second commerical diet with higher fat content. The present paired studies confirmed that in the same animals, the higher fat diet stimulated bile volume and biliary lipid secretion. On the higher fat diet there was no change in bile salt concentration or in pool size, the greater secretion rate being due to a greater bile volume and to more frequent circulations of the bile salt pool. Since the major differences between the two commercial diets were in fat and calories, the effect of supplementing the low fat diet with individual additives of olive, corn and medium chain triglyceride oil and with 40% dextrose was examined in further paired studies. In six monkeys, supplements of mono-unsaturated olive oil had no effect on biliary lipid secretion, but in seven animals given comparable doses of polyunsaturated corn oil, there was a significant increase in biliary bile salt, phospholipid and cholesterol output. The addition of medium chain triglyceride oil or of 250 ml of 40% dextrose did not change biliary lipid secretion rates. None of the dietary manipulations significantly altered cholesterol solubility in bile. In monkeys where hepatic bile salt synthesis had been maximally induced by partial biliary diversion (33.3% interruption of the EHC), corn oil no longer increased bile salt secretion suggesting that the stimulatory effect of corn oil was due to stimulation of bile salt synthesis. It is postulated that the increased turnover of bile salts seen after feeding corn oil, with a resultant acceleration in cholesterol catabolism, may explain, at least in part, the hypocholesterolaemic action of polyunsaturated fat.     

Bile acid and phospholipid fatty acid composition in bile of patients with cholesterol and pigment gallstones.

It has been previously reported that patients with cholesterol gallstones have increased biliary deoxycholate and arachidonate content as compared with normal subjects without gallstones. Increased biliary deoxycholate and arachidonate content might be a primary factor in the pathogenesis of cholesterol gallstones or merely an epiphenomenon due to the presence of gallstones. We therefore compared biliary bile acid composition in 46 patients with cholesterol gallstones and 22 patients with pigment stones. In addition, biliary phospholipid fatty acid composition was determined in 44 of these patients (30 cholesterol and 14 pigment stone patients). No significant differences were detected. In particular, the percentage deoxycholic acid (mean +/- SD: 20.3 +/- 8.8% and 21.5 +/- 10.9% respectively) and the percentage arachidonic acid (4.4 +/- 2.0% and 4.5 +/- 2.2%, respectively) were very similar. A significant correlation between age and biliary cholesterol saturation index was found only for the group of patients with pigment stones (R = 0.52, p less than 0.02). In conclusion, the present study does not support a primary role for increased biliary deoxycholic acid or arachidonic acid in the pathogenesis of cholesterol gallstones.     

Effects of hypothyroidism and cholesterol feeding on the clearance of chylomicron remnants in vivo and by rat hepatocyte monolayers

Abstract. The hepatic catabolism of chylomicron remnants in normal rats and in hypothyroid rats which were either normocholesterolaemic or made hypercholesterolaemic by feeding cholesterol and cholic acid was studied in vivo and in hepatocyte monolayers.
In vivo , the clearance of injected chylomicron remnants labelled with either cholesteryl- or retinyl ester was delayed in the hypercholesterolaemic hypothyroid rats, but not in normocholesterolaemic hypothyroid rats.
Cholesteryl-ester-rich hepatocytes from hypercholesterolaemic hypothyroid rats took up remnants less efficiently than did normal hepatocytes. Hepatocytes from normocholesterolaemic hypothyroid rats had a lower cholesteryl ester content and took up remant particles to a greater degree than did normal hepatocytes. When normal hepatocytes were incubated with hypercholesterolaemic serum or with mevalonolactone, which increased cell cholesteryl ester content, there was a slight suppression of remnant uptake. Also, addition of triiodothyronine to hepatocyte monolayers suppressed rather than increased uptake of chylomicron remants in hepatocytes.
Thus, the study suggests that chylomicron remnant uptake by the liver is not inhibited by hypothyroidism per se but by the cholesterol accumulation in hepatocytes that is the consequence of cholesterol and bile acid feeding of the rats. Although the cholesteryl ester content in hepatocytes seems to determine remnant uptake, the regulation of uptake does not seem to be as effective as that of the LDL receptor in extrahepatic cells.     

Hepatobiliary cholesterol transport is not impaired in Abca1-null mice lacking HDL

The ABC transporter ABCA1 regulates HDL levels and is considered to control the first step of reverse cholesterol transport from the periphery to the liver. To test this concept, we studied the effect of ABCA1 deficiency on hepatic metabolism and hepatobiliary flux of cholesterol in mice. Hepatic lipid contents and biliary secretion rates were determined in Abca1(-/-), Abca1(+/-), and Abca1(+/+) mice with a DBA background that were fed either standard chow or a high-fat, high-cholesterol diet. Hepatic cholesterol and phospholipid contents in Abca1(-/-) mice were indistinguishable from those in Abca1(+/-) and Abca1(+/+) mice on both diets. In spite of the absence of HDL, biliary secretion rates of cholesterol, bile salts, and phospholipid were unimpaired in Abca1(-/-) mice. Neither the hepatic expression levels of genes controlling key steps in cholesterol metabolism nor the contribution of de novo synthesis to biliary cholesterol and bile salts were affected by Abca genotype. Finally, fecal excretion of neutral and acidic sterols was similar in all groups. We conclude that plasma HDL levels and ABCA1 activity do not control net cholesterol transport from the periphery via the liver into the bile, indicating that the importance of HDL in reverse cholesterol transport requires re-evaluation.     

Dietary fish oil-induced changes in intrahepatic cholesterol transport and bile acid synthesis in rats.

Hepatic cholesterol metabolism was studied in rats fed purified diets supplemented (9% wt/wt) with either fish oil (FO) (n-3 fatty acids) or corn oil (CO) (n-6 fatty acids) for 4 wk. Rats were equipped with permanent catheters in heart, bile duct, and duodenum to allow studies under normal feeding conditions. [3H]-cholesteryl oleate-labeled small unilamellar liposomes, which are rapidly endocytosed by hepatocytes, were intravenously injected to label intrahepatic cholesterol pools, and plasma and bile were collected. FO as compared to CO induced a lowering of plasma cholesterol levels by 38% and of triglyceride levels by 69%. This reduction in plasma lipids in FO rats was accompanied by: (a) an increased bile acid pool size (28%); (b) a fourfold increase in the ratio cholic acid/chenodeoxycholic acid in bile; (c) increased biliary excretion of cholesterol (51%); (d) accelerated excretion of endocytosed free cholesterol into bile; (e) accelerated incorporation of endocytosed cholesterol in bile acids; (f) a significant increase in the bile acid-independent fraction of bile flow; and (g) a threefold increase in hepatic alkaline phosphatase activity. The results show that FO induces changes in transport and metabolic pathways of cholesterol in the rat liver, which result in a more rapid disposition of plasma-derived cholesterol into the bile.     

Inappropriate hepatic cholesterol synthesis expands the cellular pool of sterol available for recruitment by bile acids in the rat.

These studies test the hypothesis that a major determinant of excessive biliary cholesterol secretion is a level of hepatic sterol synthesis that is inappropriately high relative to the needs of the liver cell for preserving cholesterol balance. Biliary cholesterol secretion was measured in vivo in two models after loading the hepatocyte with sterol by two different mechanisms. In the first model, cholesterol was delivered physiologically to the liver in chylomicron remnants. This resulted in a sixfold increase in cholesteryl ester content and marked suppression of cholesterol synthesis, but biliary cholesterol secretion remained essentially constant. In the second model, 3-hydroxy-3-methyl-glutaryl CoA reductase levels in the liver were markedly increased by chronic mevinolin (lovastatin) administration. Withdrawal of the inhibitor resulted in a sudden fivefold increase in the rate of sterol synthesis in the liver of the experimental animals that was inappropriately high for cellular needs. This excessive synthesis, in turn, was accompanied by a fivefold increase in the cholesteryl ester content, enrichment of microsomal membranes with cholesterol and, most importantly, by a threefold increase in the rate of biliary sterol secretion. As the rate of sterol synthesis gradually returned to normal over 48 h, the cholesterol ester content, the lipid composition of the microsomal membranes, and rate of cholesterol secretion into bile also returned to baseline values. These results further support the concept of functional compartmentalization of cholesterol in the hepatocyte. Derangements that cause an inappropriately high rate of sterol synthesis in the endoplasmic reticulum may lead to an expansion of that pool of cholesterol that is recruitable by bile acids and, hence, to greater situation of the bile.     

Failure of bile acids to control hepatic cholesterogenesis: evidence for endogenous cholesterol feedback

Studies were undertaken to define the role of bile acids in the control of hepatic cholesterogenesis from acetate. Both biliary diversion and biliary obstruction increase the rate of sterol synthesis by the liver 2.5- to 3-fold. After biliary diversion, however, the bile acid content of the liver is decreased, whereas after biliary obstruction, it is markedly increased. Thus, there is no relationship between the tissue content of bile acid and the rate of hepatic cholesterol synthesis. Furthermore, restoration of the enterohepatic circulation of bile acid in animals with biliary diversion fails to prevent the rise in synthetic activity seen after this manipulation. These data indicate that bile acid plays no direct inhibitory role in the regulation of cholesterol synthesis by the liver.Other experiments were therefore undertaken to evaluate the possibility that changes in cholesterogenic activity observed after manipulation of the enterohepatic circulation of bile acid actually are the result of changes in the enterolymphatic circulation of cholesterol. In support of this thesis it was found that intestinal lymphatic diversion causes the same specific enhancement of cholesterol synthetic activity as biliary diversion and that both of these operative procedures increase enzymatic activity at the step mediated by beta-hydroxy-beta-methyl glutaryl reductase. Furthermore, the increase in the rate of sterol synthesis by the liver seen in animals with biliary diversion can be prevented by the infusion of approximately 7 mg of cholesterol/24 hr in the form of chylomicrons. This is an amount of cholesterol circulating normally in the enterolymphatic circulation of the intact rat.These results indicate that bile acid plays no direct role in the control of hepatic cholesterogenesis, but rather, it is the enterohepatic circulation of endogenous cholesterol that determines directly the rate at which cholesterol is synthesized by the liver.