Effects of dexamethasone and linoleic acid on hepatic secretion of biliary lipids and anionic polypeptide factor: In vivo and in vitro studies.

Synthetic glucocorticoids, such as dexamethasone, and diets enriched with unsaturated fatty acids have been shown to stimulate hepatic bile salt synthesis. This fact led us to investigate the effects of dexamethasone and linoleic acid supplementation on bile secretion. Cholesterol (Ch) and phospholipid secretions are bile acid dependent. Ch and phospholipid in bile are also highly bound to a small apoprotein, the anionic polypeptide factor (APF). In bile, APF may play a physiological role in stabilizing cholesterol:phospholipid vesicles and might also be important in the regulatory process of bile lipid secretion. In order to study the factors influencing bile secretion, the biliary secretion rates of bile lipids and APF were experimentally modulated in perfused rat liver (PRL) and HepG2 cells. As expected, dexamethasone induced an increase in the biliary secretion rate of bile salts (BS) in the two models (PRL: 34 up to 67 nmol/l/min/g liver; HepG2 cells: 234% vs. 100% in controls). The bile secretion rates for phospholipids (PRL: from 5 down to 1.5 nmol/l/min/g liver; HepG2 cells: 93 vs. 100% in controls) and APF (PRL: from 0.34 down to 0.12 microg/l/min/g liver; cells: 86 vs. 100% in controls) rapidly decreased independently from those of BS. The data from experimental cell models supplemented with linoleic acid indicated a correlation between the BS and APF levels (APF: 71 and 63%; BS: 161 and 197% vs. 100% in controls). The phospholipid level was regulated independently from that of APF and BS and increased (106 and 111% vs. 100% in controls), while Ch remained nevertheless unchanged. Our data showed that dexamethasone induced changes in bile and that linoleic acid clearly impaired the regulation exerted by the dexamethasone on bile lipids.

Modulating effects of bile salt hydrophobicity on bile secretion of the major protein of the bile lipoprotein complex.

Bile lipids are secreted in association with a newly identified major apoprotein called anionic polypeptide fraction-calcium binding protein (APF-CBP), which is synthesized in the hepatocytes and has been detected in both bile and plasma and characterized. The secretion of the lipids in bile depends both on the concentration and the hydrophobicity of the bile salts (BS) secreted. The present study was undertaken to determine whether the synthesis and the secretion of APF-CBP are similarly regulated by BS, using two methods. The synthesis and secretion of labelled, newly synthesized APF-CBP by isolated rat hepatocytes were monitored by solid-phase immunoassay. For this purpose, hepatocytes were incubated with either glycodeoxycholate (GDC) or taurocholate (TC). The synthesis and secretion of labelled, newly synthesized APF-CBP by perfused rat liver were measured by immunological enzyme-linked assay (ELISA) upon perfusing the liver with either GDC or TC. We found that (i) the synthesis and the secretion of APF-CBP were increased during either TC or GDC perfusion, but the increase was more pronounced with TC; (ii) in GDC perfusion the APF-CBP levels measured were more closely related to the levels of bile salts and not to phospholipid levels, (iii) when the two bile salts were perfused in reverse order, i.e., first GDC and then TC, the secretion of APF-CBP in bile decreased when GDC was perfused, but increased when TC was perfused. Similar results were obtained in experiments with isolated hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Monensin action on the Golgi complex in perfused rat liver: evidence against bile salt vesicular transport.

Several studies suggest that bile salts are transported from the basolateral to the canalicular membrane of hepatocytes by a vesicular pathway, possibly in part via the Golgi complex. To test this hypothesis, the present study examined, in the perfused rat liver, the influence of the Na+ ionophore monensin on the biliary secretion of taurocholate and biliary lipids. The effects of the drug have been checked by the study of the ultrastructural modifications of the Golgi complex, secretion of horseradish peroxidase, and bile salt uptake. An infusion of monensin (1, 3, or 5 mumol/L) into the liver induced considerable swelling of the Golgi complex within 5 minutes. After a bolus injection of horseradish peroxidase during monensin infusion, the biliary secretion of the protein was delayed (1 mumol/L monensin) and markedly reduced (5 mumol/L monensin). Bile salt uptake was virtually unchanged except with 5 mumol/L monensin. This suggests that monensin has the same effects on the subcellular traffic in the perfused liver as in cultured cells. After a bolus injection of taurocholate (0.25, 5.0, or 8.5 mumol/100 g body wt) during monensin infusion, the pattern of biliary secretion of the bile salt was identical to that of controls. During continuous infusion of taurocholate, a 10-minute monensin infusion (1 or 3 mumol/L) had no effect on the biliary secretion of taurocholate and on the secretion of lecithin and cholesterol induced by taurocholate. High concentrations (5 mumol/L) or prolonged infusions (20 minutes) of monensin decreased the biliary secretion of bile salts but corresponded to a marked decrease of taurocholate uptake. In summary, the Na+ ionophore monensin altered the Golgi complex and the vesicular transport of horseradish peroxidase, whereas taurocholate biliary secretion was not influenced unless taurocholate biliary secretion was not influenced unless taurocholate uptake by the liver was markedly decreased. It may be concluded that taurocholate and biliary lipid secretion, under these conditions, does not depend essentially on pathways involving acidic transporting vesicles and particularly the trans-Golgi complex.

[Intracellular lumen in cultured hepatocytes of rats. Study of an original cytoplasmic infrastructure]. / La lumière intracellulaire dans les hépatocytes de rat en culture. Etude d'une infrastructure cytoplasmique originale.

A few hours after plating, isolated rat hepatocytes reassociate into clusters and differentiate intercellular cavities bordered by junctional complexes. These structures show a great resemblance to bile canaliculi seen in vivo. Intracellular lumens surrounded by microvilli are observed in the cytoplasm of some cultured hepatocytes. The formation of these structures, which contain an osmiophilic substance, probably results from modifications in the functioning of the secretory apparatus. It can be speculated that these intracellular lumens may contribute to the formation of new canaliculi differentiated between reassociated cells.

Influence of lipid-rich diets on intestinal cholesterol uptake.

The purpose of this work was to determine whether or not cholesterol transport by intestinal brush border is influenced by fat-enriched diets and therefore plays a role in the regulation of cholesterol absorption. This study was carried out on rats divided into three groups according to diet: (1) control with a diet containing 1.2% cholesterol (diet T), (2) diet T plus 28% saturated fat (lard) and (3) diet T plus 28% polyunsaturated fat (corn oil). Uptake of cholesterol and oleic acid from defined mixed micellar solutions was studied on two experimental models: everted sacs and brush border vesicles of the intestinal membrane. In vivo cholesterol absorption was measured by the dual isotope plasma ratio method of Zilversmit. Fat-enriched diets decreased both in vivo cholesterol absorption and in vitro cholesterol uptake without any specific effect of unsaturated fats. This suggests that the mechanisms involved in the transport of cholesterol across the brush border membrane may be rate-limiting for cholesterol absorption. Oleate and butyrate uptakes, in contrast, were unaffected by the fat content of the diet.

Intestinal cholesterol and oleic acid uptake from solutions supersaturated with lipids.

To test the role of nonmicellar phases in lipid absorption, intestinal uptake of fatty acids and cholesterol has been studied in vitro from supersaturated and micellar solutions. The micellar solubility limit at equilibrium was established for cholesterol and oleate/monoolein (2:1) at pH 6.7 with 10 mM taurocholate. Uptake by rat intestinal everted sacs was measured during incubation of 5 min. Cholesterol uptake increased linearly with the cholesterol content of micellar or supersaturated solutions up to a supersaturation of 150%. Oleate uptake, by contrast, remained essentially the same from either saturated or supersaturated (130-280%) mixtures. The difference between cholesterol and oleate uptake rates is explained by their distinct effects on micellar size, which is unchanged by cholesterol supersaturation but is increased by oleate. Solutions largely supersaturated (280%) with oleate-monoolein are polydisperse and contain viscous isotropic and paracrystalline phases similar to those observed during lipid absorption. These results suggest that, in the presence of such solutions, uptake occurs from both the micellar saturated and nonmicellar supersaturated phases.

Intestinal cholesterol uptake: comparison between mixed micelles containing lecithin or lysolecithin.

The aim of our study was to define the mechanism by which cholesterol uptake is inhibited by lecithin but not by lysolecithin. The work compared the cholesterol uptake by everted rat jejunal sacs from bile salt-lecithin-cholesterol or bile salt-lysolecithin-cholesterol micelles. The micellar size and the cholesterol saturation were measured. The size or molecular weight increases when the lecithin concentration rises, and the cholesterol uptake decreases and leads to zero when the micelles contain more than 30% lecithin. The size of bile salt-lysolecithin-cholesterol micelles is smaller than that of lecithin micelles in comparable molar ratios. Consistent with this result is the fact that, for a given phospholipid concentration, cholesterol uptake is greater in the presence of lysolecithin than in the presence of lecithin. The diffusion rate of the micelles through the unstirred water layer decreases when micellar size increases. However, the comparison of uptakes from lecithin or lysolecithin micelles similar in size and in cholesterol saturation showed that the cholesterol uptake is still lower for lecithin micelles. This shows that with larger micelles some factor other than micellar size and cholesterol content of the micelles is important. We observe that lysolecithin absorption is 15-fold greater than lecithin absorption. We suggest that lysolecithin absorption results in a rapid supersaturation with cholesterol leading to cholesterol absorption.

Mixed micelle properties and intestinal cholesterol uptake.

The solubilizing powers of taurocholate, taurochenodeoxycholate and tauroursodeoxycholate for monoolein and cholesterol, and the size of the bile salt-monoolein-cholesterol micelles have been determined. For the three bile salt species, the micellar size depends on the saturation with monoolein. As a result, for a given bile salt to monoolein ratio, the taurochenodeoxycholate micelles are smaller than those of taurocholate and both are smaller than those of tauroursodeoxycholate. Intestinal cholesterol uptake has been studied in vitro as a function of the micellar size and the saturation degree with cholesterol. For a given bile salt to monoolein ration and 1) for low cholesterol concentrations, taurocholate leads to the greatest rates of uptake ; 2) for high cholesterol content, taurochenodeoxycholate induces the largest uptake. The specific micellar characteristics of the tauroursodeoxycholate micelles clearly demonstrate why this bile salt is of so little help in the intestinal uptake of cholesterol.

Intestinal cholesterol uptake from mixed micelles. In vitro effects of taurocholate, taurochenodeoxycholate and tauroursodeoxycholate.

Cholesterol uptake by everted rat jejunal sacs is lower from mixed micelles containing tauroursodeoxycholate than from those with taurocholate or taurochenodeoxycholate. This occurs in spite of a greater saturation with cholesterol of tauroursodeoxycholate micelles as measured by equilibrium solubility studies. The results suggest that cholesterol saturation of solutions containing tauroursodeoxycholate is overestimated when calculated with reference to solubility in micellar form.

Comparative effects of cholic, chenodeoxycholic, and ursodeoxycholic acids on micellar solubilization and intestinal absorption of cholesterol.

Cholesterol absorption was studied in mice receiving cholic, chenodeoxycholic, or ursodeoxycholic acids (0.2% of the diet) for 2 months. Cholesterol absorption was greater with cholic acid (79%) than with chenodeoxycholic acid feeding (60%) and the lowest levels were observed during ursodeoxycholic acid feeding (37%). Under the three diets, bile acid pool and bile acid secretion were not different. Biliary cholesterol secretion was increased by cholic acid. The bile acid fed represents at least 80% of total bile acids. Micellar solubilization of oleic acid and cholesterol in the presence of each tauro-conjugated bile salt (10 mM) was determined in vitro by the coprecipitation method. Whatever the pH conditions, taurochenodeoxycholate solubilized significantly more cholesterol and more oleic acid than taurocholate. Tauroursodeoxycholate had the poorest detergent properties for both lipids. The differences between the three bile salts for cholesterol solubilization were enlarged by lowering pH and by high oleic acid concentration. Therefore the decrease in cholesterol absorption observed during ursodeoxycholic acid feeding could be explained by the poor detergent properties of this bile salt species. On the other hand, there is no relationship between the detergent properties of taurochenodeoxycholate and taurocholate and their effects on cholesterol absorption in mice. These results suggest that, in this particular case, micellar solubilization is not the rate limiting step in cholesterol absorption.

[Effect of cholic acid and chenodesoxycholic acid on biliary secretion in mice. Effect of the addition of beta-sitosterol]. / Action de l'acide cholique et de l'acide chénodesoxycholique sur la sécrétion biliaire de la souris. Effet de l'addition du beta-sitostérol.

Five groups of 20 mice received for 4 months one of the following diets: T, standard diet (T); a, T + cholic acid (0.2%); b, T + cholic acid (0.2%) + beta-sitosterol (2%); c, T + chenodeoxycholic acid (0.2%); d, T + chenodeoxycholic acid (0.2%) + beta-sitosterol (2%). After this time, the cholesterol intestinal absorption and the biliary secretion of lipids were measured. The biliary secretion of cholesterol, the total hepatic cholesterol (23 mg/g liver dry weight), and the intestinal absorption of cholesterol (90% administered dose) were higher in mice fed with cholic acid than in mice fed with chenodeoxycholic acid (hepatic cholesterol, 9.6 mg/g liver dry weight; absorption, 65% administered dose). The addition of beta-sitosterol to the diet supplemented with cholic acid decreased the cholesterol intestinal absorption and the biliary secretion of cholesterol so that both became similar to that obtained with chenodeoxycholic acid. These results indicate that in mice, as in man, cholic acid elicits a higher cholesterol biliary secretion than chenodeoxycholic acid. In this experimental model, the distinct effect on the biliary cholesterol of these two bile salts is due to their specific effects on the intestinal absorption of cholesterol.

Distinct effects of three bile salts on cholesterol solubilization by oleate-monoolein-bile salt micelles.

Micellar cholesterol solubilities in bile salt-monoolein-oleic acid systems have been determined. Whatever the bile salt/oleyl compounds ratio, taurochenodeoxycholate solubilizes more cholesterol than taurocholate and much more than tauroursodeoxycholate. At pH 6.7, the cholesterol solubility limit is about the same with either oleate or monoolein. Cholesterol solubility falls in oleate-bile acid mixtures as the pH is raised. The capacity for supersaturation with cholesterol is greater for bile salt-monoolein than for bile salt-oleate micelles. For the latter it decreases as pH increases.

Action of three bile acids on hepatic and intestinal cholesterogenesis in the rat.

Incorporation of [1(14)-C] acetate into cholesterol by subcellular particles from the liver and the small intestine of rats with a biliary diversion and a duodenal perfusion of sodium taurocholate, taurochenodeoxycholate or taurodehydrocholate, was studied in vitro. In the liver, taurochenodeoxycholate prevented the increase of cholesterol synthesis induced by biliary drainage. Taurocholate had no action on cholesterol synthesis at any time, day or night. Intestinal synthesis of cholesterol was reduced by taurocholate and taurochenodeoxycholate but was not modified by taurodehydrocholate infusion.

Ultrastructural alterations and modifications of nuclear RNA of rat liver by the combined action of thioacetamide and aflatoxin.

When aflatoxin is administered to thioacetamide-treated rats, the synthesis of nuclear RNA not only stops but the RNA that had accumulated in the nuclei by thioacetamide action disappears, probably by degradation "in situ" as none appears in the cytoplasm. Morphologically, the lesions provoked by aflatoxin add to those caused by thioacetamide. In the gigantic nucleoli that develop upon exposure to thioacetamide, aflatoxin provokes atypical segregations that result in the formation of larger and larger spaces in the nucleoli.

Diurnal changes of bile lipid concentration in hepatic bile of cholecystectomized man.

Diurnal variations of bile lipid concentration were studied in ten patients with a tube in the main bile duct following a cholecystectomy. 5-6 bile samples per 24 h were collected from each patient during 3-40 days. The enterohepatic cycle was not significantly modified since total bile samples did not exceed 40 ml/day. Significant diurnal variations were observed in cholesterol concentration. Changes in lecithin concentration seemed to be similar in seven patients but did not reach the level of significance in any individual patient. Maximal values were observed between 4 and 8 a.m. and minimal values at 4 p.m. Bile salt concentration varied without any circadian periodicity. Mean bile lipid concentration was calculated for each patient. The patients with highest cholesterol concentrations have also the highest mean lecithin concentration. Mean bile salt concentration does not differ much from one patient to another.