Regulation of gallbladder cholesterol concentration in the hamster. Role of hepatic cholesterol level

The goal of this investigation was to determine how alterations in hepatic cholesterol metabolism influence the cholesterol content of gallbladder bile in hamsters. Although the rate of hepatic cholesterol synthesis was varied over 600-fold, there was no direct relationship between the rate of cholesterol synthesis and the cholesterol content of gallbladder bile. However, expansion of the hepatic cholesterol pool by 42-fold resulted in an 11-fold increase in gallbladder bile cholesterol. Examination of four subfractions of the hepatic cholesterol pool revealed that the cholesterol content of gallbladder bile was most consistently correlated with the free cholesterol level in both hepatic tissue and hepatic microsomes from all experimental groups. In most groups of animals in which gallbladder bile cholesterol was increased, plasma lipoprotein cholesterol levels were also increased. It was concluded that in hamsters, under these experimental conditions, changes in the cholesterol content of gallbladder bile were directly related to alterations in cholesterol content of the liver and most closely related to alterations in the free cholesterol content of that tissue.     

Leptin promotes biliary cholesterol elimination during weight loss in ob/ob mice by regulating the enterohepatic circulation of bile salts

Leptin administration to obese C57BL/6J (ob/ob) mice results in weight loss by reducing body fat. Because adipose tissue is an important storage depot for cholesterol, we explored evidence that leptin-induced weight loss in ob/ob mice was accompanied by transport of cholesterol to the liver and its elimination via bile. Consistent with mobilization of stored cholesterol, cholesterol concentrations in adipose tissue remained unchanged during weight loss. Plasma cholesterol levels fell sharply, and microscopic analyses of gallbladder bile revealed cholesterol crystals as well as cholesterol gallstones. Surprisingly, leptin reduced biliary cholesterol secretion rates without affecting secretion rates of bile salts or phospholipids. Instead, cholesterol supersaturation of gallbladder bile was due to marked decreases in bile salt hydrophobicity and not to hypersecretion of biliary cholesterol per se, such as occurs in humans during weight loss. In addition to regulating bile salt composition, leptin treatment decreased bile salt pool size. The smaller, more hydrophilic bile salt pool was associated with substantial decreases in intestinal cholesterol absorption. Within the liver, leptin treatment reduced the activity of 3-hydroxy-3-methylglutaryl-CoA reductase, but it did not change activities of cholesterol 7alpha-hydroxylase or acyl-CoA:cholesterol acyltransferase. These data suggest that leptin regulates biliary lipid metabolism to promote efficient elimination of excess cholesterol stored in adipose tissue. Cholesterol gallstone formation during weight loss in ob/ob mice appears to represent a pathologic consequence of an adaptive response that prevents absorption of biliary and dietary cholesterol.     

Phenotypic characterization of lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice. Pathophysiology Of biliary lipid secretion.

The inbred C57L strain but not the AKR strain of mice carry Lith genes that determine cholesterol gallstone susceptibility. When C57L mice are fed a lithogenic diet containing 15% fat, 1% cholesterol, and 0.5% cholic acid, gallbladder bile displays rapid cholesterol supersaturation, mucin gel accumulation, increases in hydrophobic bile salts, and rapid phase separation of solid and liquid crystals, all of which contribute to the high cholesterol gallstone prevalence rates (D. Q-H. Wang, B. Paigen, and M. C. Carey. J. Lipid Res. 1997. 38: 1395;-1411). We have now determined the hepatic secretion rates of biliary lipids in fasting male and female C57L and AKR mice and the intercross (C57L x AKR)F(1) before and at frequent intervals during feeding the lithogenic diet for 56 days. Bile flow and biliary lipid secretion rates were measured in the first hour of an acute bile fistula and circulating bile salt pool sizes were determined by the "washout" technique after cholecystectomy. Compared with AKR mice, we found that i) C57L and F(1) mice on chow displayed significantly higher secretion rates of all biliary lipids, and larger bile salt pool sizes, as well as higher bile salt-dependent and bile salt-independent flow rates; ii) the lithogenic diet further increased biliary cholesterol and lecithin outputs, but bile salt outputs remained constant. Biliary coupling of cholesterol to lecithin increased approximately 30%, setting the biophysical conditions necessary for cholesterol phase separation in the gallbladder; and iii) no gender differences in lipid secretion rates were noted but male mice exhibited significantly more hydrophobic bile salt pools than females.We conclude that in gallstone-susceptible mice, Lith genes determine increased outputs of all biliary lipids but promote cholesterol hypersecretion disproportionately to lecithin and bile salt outputs thereby inducing lithogenic bile formation.     

Mathematical model of biliary lipid secretion: a quantitative analysis of physiological and biochemical data from man and other species

We propose a simple mathematical model to account for the coupling of secretion rates of bile salts, lecithin, and cholesterol into bile. The model assumes that: 1) molecules of "biliary" lecithin and cholesterol enter a functional compartment located in the endoplasmic reticulum of the hepatocyte from which they are secreted into bile, and in the case of cholesterol, also catabolized to bile salts; 2) the rates at which lecithin and cholesterol enter the "secretory" compartment are regulated independently by feedback loops that control their synthesis and/or uptake; 3) lecithin secretion is coupled by an unknown transport mechanism, possibly micellar or vesicular, to the flux of bile salts passing through the compartment; 4) cholesterol secretion is coupled by a similar mechanism to lecithin secretion and not to bile salt secretion directly; and 5) bile salt synthesis is proportional to the cholesterol content of the compartment. The model predicts that in the steady state the dependences, lecithin secretion vs bile salt secretion; cholesterol secretion vs lecithin secretion; and cholesterol secretion vs bile salt secretion, will all have the form of rectangular hyperbolae. Four independent parameters related to the postulated mechanisms of biliary lipid synthesis, uptake, and transport determine the quantitative features of these hyperbolae. These four "secretion parameters" also determine how the biliary lipid composition of hepatic and "fasting" gallbladder bile varies with bile salt secretion rate. A quantitative analysis of biochemical and physiological data on biliary lipid secretion in rat, dog, and man confirms the general predictions of the model. Deductions of the secretion parameters are made for each species and are compared with other relevant data on biliary lipid metabolism. From this analysis, we offer new insights into: i) the species differences in biliary lipid secretion and bile composition; ii) the influence of obesity on biliary lipid secretion in man; and iii) the causes of cholesterol super-saturation in fasting gallbladder bile.     

Feedback regulation of bile acid biosynthesis in the rat

The hepatic biosynthesis of bile salts in the rat has been shown to be controlled homeostatically by the quantity of bile salt returning to the liver via the portal circulation. The feedback mechanism was demonstrated in two kinds of experiments. In the first, rats with bile fistulas were infused intraduodenally with sodium taurocholate 12 hr after surgery. If the rate of infusion was greater than 10 mg per 100 g rat per hr, the increase in bile acid output normally observed in bile fistula rats was prevented. In the second type of experiment, the rats were infused with taurocholate 48-72 hr after biliary diversion, when bile acid output had reached a maximal value. Provided the rate of infusion exceeded 10 mg per 100 g rat per hr, bile acid secretion returned to the low levels observed in intact rats. Previous attempts to demonstrate the feedback control have been unsuccessful because too little bile salt was infused. The taurocholate pool of the experimental animals was measured as approximately 15 mg per 100 g rat; it was calculated from this and the above results that this pool circulated 10-13 times daily.     

Control of Bile Salt Synthesis

THE regulation of bile salt synthesis in man is still poorly understood. The human liver synthesizes a trihydroxy and a dihydroxy bile acid, cholic and chenodeoxycholic acid, each conjugated with glycine or taurine. These, together with deoxycholic acid, a dihydroxy bile acid which results from the 7-α dehydroxylation of cholic acid by intestinal bacteria, constitute approximately 98% of the bile salt pool which circulates enterohepatically during digestion of a meal¹. Lindstedt found that the composition of the bile salt pool varies little from day to day in any one individual² and this has also been our experience on analysis of 48 bile samples collected on different days from 16 volunteers. However, the evidence that bile salt synthesis is regulated by a negative feedback mechanism is scanty³ apart from the increased synthesis that follows interruption of the enterohepatic circulation^4–6.     

Enterohepatic circulation of bile salts in farnesoid X receptor-deficient mice: efficient intestinal bile salt absorption in the absence of ileal bile acid-binding protein

The bile salt-activated farnesoid X receptor (FXR; NR1H4) controls expression of several genes considered crucial in maintenance of bile salt homeostasis. We evaluated the physiological consequences of FXR deficiency on bile formation and on the kinetics of the enterohepatic circulation of cholate, the major bile salt species in mice. The pool size, fractional turnover rate, synthesis rate, and intestinal absorption of cholate were determined by stable isotope dilution and were related to expression of relevant transporters in the livers and intestines of FXR-deficient (Fxr-/-) mice. Fxr-/- mice showed only mildly elevated plasma bile salt concentrations associated with a 2.4-fold higher biliary bile salt output, whereas hepatic mRNA levels of the bile salt export pump were decreased. Cholate pool size and total bile salt pool size were increased by 67 and 39%, respectively, in Fxr-/- mice compared with wild-type mice. The cholate synthesis rate was increased by 85% in Fxr-/- mice, coinciding with a 2.5-fold increase in cholesterol 7alpha-hydroxylase (Cyp7a1) and unchanged sterol 12alpha-hydroxylase (Cyp8b1) expression in the liver. Despite a complete absence of ileal bile acid-binding protein mRNA and protein, the fractional turnover rate and cycling time of the cholate pool were not affected. The calculated amount of cholate reabsorbed from the intestine per day was approximately 2-fold higher in Fxr-/- mice than in wild-type mice. Thus, the absence of FXR in mice is associated with defective feedback inhibition of hepatic cholate synthesis, which leads to enlargement of the circulating cholate pool with an unaltered fractional turnover rate. The absence of ileal bile acid-binding protein does not negatively interfere with the enterohepatic circulation of cholate in mice.     

Effects of cholecystectomy upon bile salt kinetics in the guinea pig

The effects of cholecystectomy upon bile salt kinetics were studied in normal guinea pigs. After cholecystectomy, bile salt pool size decreased, fractional daily turnover rate increased, and the rate of bile salt synthesis was unchanged. These data indicate that an increased frequency of bile salt enterohepatic cycling is sufficient to produce alterations in bile salt kinetics. Abnormalities of bile salt synthesis need not be present in order for a reduction in pool size to occur.     

Female sex steroid induced epithelial changes in the gallbladder of the ovariectomized Syrian hamster.

Ovariectomized Syrian hamsters treated by female sex steroids during a 1-month period show gallbladder surface epithelial changes in the fundic area consistent with apical bulging and decapitations of the epithelial cells. These events were detected in the infundibulum and the fundic or body regions of estrogen- and estrogen+progesterone-treated hamsters. In control hamsters, these events were restricted to the region in the vicinity of the bile duct. Following steroid treatment, intraluminal deposits detected resembled Ca-bilirubinate deposits described in previous studies while decapitations are similar to endometrial epithelium changes associated with hormonal physiological changes or treatments. Moreover some small electron-dense deposits are comparable to those found in human cholesterol gallstones. This report indicates that, besides an alteration in bile composition, cell fragments originating from the surface epithelium of the bile duct and/or of the gallbladder mucosal epithelium could participate in gallstone nucleation.     

Effects of plasma cholesterol lowering agents on hepatobiliary lipid metabolism and cholesterol turnover in the rhesus monkey.

The effects of clofibrate, cholestyramine, and neomycin on hepatobiliary lipid metabolism were studied in adult rhesus monkeys in metabolic steady state with intact but exteriorized enterohepatic circulations. Clofibrate (30 mg/kg, id) had no effect on lipid secretion while cholestyramine (150 mg/kg, id) decreased biliary cholesterol secretion rate from 0.19 +/- 0.03 to 0.13 +/- 0.02 mmol/24 h, p less than 0.05. Neomycin (30 mg/kg, id) decreased bile flow from 216 +/- 10 to 191 +/- 7mL/24 h, p less than 0.05, and tended only to decrease bile salt and phospholipid secretion rates. Cholestyramine decreased cholesterol composition from 1.81 +/- 0.22 to 1.30 +/- 0.22 mol %, p less than 0.05, while clofibrate and neomycin had insignificant effects. Cholestyramine and neomycin decreased bile salt pool size from 1 +/- 0.1 to 0.77 +/- 0.15 and from 1.45 +/- 0.16 to 1.13 +/- 0.21 mmol, p less than 0.05, respectively, while clofibrate had no effect. Bile salt synthetic rate was increased only by cholestyramine, i.e., from 0.63 +/- 0.04 to 1.48 +/- 0.26 mmol/24 h, p less than 0.01. Concomitant cholesterol turnover studies revealed that cholestyramine increased the production rate and excretion of cholesterol in the rapidly miscible cholesterol pool and increased the transfer of cholesterol from slow to rapidly miscible pools. Neomycin, on the other hand, decreased the size of the rapidly miscible pool by decreasing production rate without affecting the size of the slowly miscible pool, while clofibrate had insignificant effects.     

Increased filamentous actin in islets of Langerhans from fasted hamsters

We measured pools of soluble and sedimentable actin in hamster islets using a new DNase I binding/immunoprecipitation assay. Islets from fed and fasted hamsters contained the same amount of actin when expressed per microgram of protein. In three experiments the sedimentable (filamentous) actin increased significantly in islets from fasted hamsters. Recovery determinations demonstrate that during fasting the sedimentable actin pool (F) is increased as the soluble (G) actin pool decreases. These results suggest that the microfilament system is affected by the metabolic state of hamsters and may be responsible, in part, for inhibiting insulin secretion during fasting.     

Variation in Hepatic Bile Composition Following Cholecystectomy in Patients with Previous Gallstones

The composition of fasting hepatic bile was analyzed in 63 samples from 8 patients following cholecystectomy to determine if bile was lithogenic in patients with previous cholesterol gallstones after removal of the gallbladder. Bile specimens were obtained from t-tubes over a 7-20 day study period following re-establishment of the enterohepatic circulation. Bile composition varied on a day to day basis in each patient. 18 of 63 samples were lithogenic according to criteria of Admirand and Small while 35 of 63 samples were lithogenic according to criteria of Hegardt and Dam. Variations in the composition of hepatic bile appeared related to changes in the excretion rate of bile acids. These studies demonstrate that hepatic bile may be lithogenic after cholecystectomy and indicate that factors other than sequestration of the bile acid pool in the gallbladder influence the enterohepatic circulation of bile acids and the lithogenicity of bile.     

Effect of high sodium chloride concentrations on the pigment content and free-radical processes in corn seedlings leaves

The effect of sodium chloride on general morphometrical parameters of seedlings, and biochemical parameters in the leaves of corn seedlings was studied. Exposure to 100 and 200 mM NaCl slowed down the growth of stem and roots, whereas 100 and 200 mM NaCl during 24 h enhanced the concentration of chlorophylls, carotenoids, anthocyans, and thiobarbituric acid reactive substances. The decrease in protein carbonyl groups was found at 24-hour exposure to 200 mM salt. The treatment during 24, 48 and 72 h to 200 mM salt increased the level of total and high molecular mass thiols, whereas low molecular mass thiol content was by 20-25% higher at 48 h exposure to all used salt concentrations. The activity of guaiacol peroxidase was higher only at 24 h exposure to 100 and 200 mM salt, and catalase--at 50 mM during 48 h. At 72-hour exposure, catalase activity was by 27 and 41% higher in seedlings, exposed to 50 and 200 mM NaCl, respectively. Therefore, it is concluded the plant exposure to 50-200 mM salt initially developed oxidative stress, inducing adaptive response--an increase in antioxidant potential and efficiency of systems of energy production. That results in plant adaptation to unfavourable conditions.     

Regulation of Bile Salt Pool Size in Man

When the enterohepatic circulation is intact the size of the bile salt pool is largely determined by the frequency of its enterohepatic circulation. This hypothesis, derived from studies of cholate kinetics in coeliac, cholecystectomy, and gall stone patients, represents the simplest interpretation of the data. A corollary is that daily cholate secretion is likely to be normal in these conditions and that therefore the propensity of bile to form cholesterol gall stones is not likely to be directly related to bile salt pool size.     

Estimation of bile acid pool sizes from their spillover into systemic blood

We have examined the possibility of assessing primary bile acid pool sizes from the spillover of the bile acids into systemic blood after intestinal exposure to the total endogenous bile acid pool; the studies were carried out in 16 healthy subjects. Bile acid spillover was calculated as the integrated area under the curve of bile acid conjugates in serum of each primary bile acid class in response to a well-defined sustained cholecystokinin-induced stimulus of the enterohepatic circulation for 55 min causing complete gallbladder emptying. Serum levels of each species of primary bile acid conjugates were measured by two specific and sensitive radioimmunoassays, one for conjugated cholate and one for conjugated chenodeoxycholate. Primary bile acid pool sizes determined with [24-14C]cholic acid and [24-14C]chenodeoxycholic acid according to Lindstedt (1957. Acta Physiol. Scand. 40:1-9) served as reference. Bile acid conjugates of both species reached a peak 70 min after the start of the cholecystokinin infusion, probably reflecting simultaneous intestinal absorption of both primary bile acids in this model. Highly significant linear correlations were found between the integrated areas under the curve and primary bile acid pool sizes, which were closer for chenodeoxycholate (n = 16, r = 0.81, P less than 0.001) than for cholate (n = 16, r = 0.74, P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuropeptide Y content in the hypothalamic paraventricular nucleus responds to fasting and refeeding in broiler chickens

To examine the neural mechanism by which hypothalamic neuropeptide Y (NPY) regulates energy homeostasis and feeding behavior in commercial broilers, we measured NPY content in several hypothalamic regions of birds that were fasted and then refed. After fasting for 48 and 72 h, body weight significantly decreased, and food intake significantly increased during the subsequent refeeding. The lost body weight was not restored to ad libitum feeding levels even after 3 days of refeeding. Plasma glucose concentration and body fat content significantly decreased and plasma non-esterified fatty acid (NEFA) concentration significantly increased after 48- and 72-h fasting. Refeeding for 24 h restored plasma metabolites and body fat content to pre-fasting levels. NPY content in the paraventricular nucleus (PVN) and infundibular nucleus significantly increased during fasting, and NPY content of the PVN was restored to pre-fasting levels after 24-h refeeding. However, there was no significant change in the NPY content of the lateral hypothalamic area during fasting or refeeding. The present results of changes in the hypothalamic NPY content during fasting and refeeding support the hypothesis that NPY plays a central role in regulation of energy homeostasis, with especially important effect on feeding behavior and body weight in broiler chickens.     

Pathogenesis of human cholesterol cholelithiasis.

The pathogenesis of cholesterol cholelithiasis in humans has been studied by means of three techniques. The cholesterol-solubilizing capacity of bile may be determined by estimation of the relative composition of the three major lipid constituents of bile. Consistent reduction in the cholesterol-carrying capacity of gallbladder bile of persons with gallstones when compared with normal subjects has not been shown. Normal subjects frequently have supersaturated bile. Secretion rates of biliary lipids have been estimated by two methods; with the method that appears to be more physiologic no change in lipid secretion rates was found in gallstone patients. Bile acid pool size has been measured by isotope dilution techniques; it is reduced in patients with gallstones. It is not clear whether this reduction is important in the pathogenesis of cholesterol cholelithiasis, for the bile acid secretion rate is normal because of an increased rate of cycling of the pool through the enterohepatic circulation. The role of the gallbladder in the genesis of cholesterol cholelithiasis may be more important than has been realized.     

Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats

Human milk contains prebiotic oligosaccharides, which stimulate the growth of intestinal bifidobacteria and lactobacilli. It is unclear whether the prebiotic capacity of human milk contributes to the larger bile salt pool size and the more efficient fat absorption in infants fed human milk compared with formula. We determined the effect of prebiotic oligosaccharides on bile salt metabolism in rats. Rats were fed a control diet or an isocaloric diet containing a mixture of galactooligosaccharides (GOS), long-chain fructooligosaccharides (lcFOS), and acidified oligosaccharides (AOS) for 3 wk. We determined synthesis rate, pool size, and fractional turnover rate (FTR) of the primary bile salt cholate by using stable isotope dilution methodology. We quantified bile flow and biliary bile salt secretion rates through bile cannulation. Prebiotic intervention resulted in significant changes in fecal and colonic flora: the proportion of lactobacilli increased 344% (P < 0.01) in colon content and 139% (P < 0.01) in feces compared with the control group. The number of bifidobacteria also increased 366% (P < 0.01) in colon content and 282% in feces after the prebiotic treatment. Furthermore, pH in both colon and feces decreased significantly with 1.0 and 0.5 pH point, respectively. However, despite this alteration of intestinal bacterial flora, no significant effect on relevant parameters of bile salt metabolism and cholate kinetics was found. The present data in rats do not support the hypothesis that prebiotics naturally present in human milk contribute to a larger bile salt pool size or altered bile salt pool kinetics.