The effect of chylomicron remnants on bile acid synthesis in cultured rat hepatocytes

The effect of chylomicron remnants on bile acid synthesis in isolated rat hepatocytes in monolayer cultures was investigated. Production of bile acids by the cells in the presence of chylomicron remnants at a cholesterol concentration of 7.8-9 nmol/ml was increased by approx. 75% after 17 h and 25% after 24 h incubation. Similar concentrations of cholesterol added to the cells in the form of chylomicrons had no significant effect on bile acid synthesis. These results suggest that cholesterol taken up in chylomicron remnants may be an important source of substrate for bile acid synthesis.     

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.     

cAMP analogues inhibit phosphatidylcholine biosynthesis in cultured rat hepatocytes

The effect of cAMP analogues on phosphatidylcholine formation via the CDP-choline pathway was investigated in cultured monolayers of rat hepatocytes. Treatment with chlorophenylthio-cAMP or the cAMP phosphodiesterase inhibitor, aminophylline, reduced the total uptake of [methyl-3H]choline by 32 and 26% (p less than 0.01), respectively. Chlorophenylthio-cAMP inhibited the incorporation of [methyl-3H]choline into phosphatidylcholine by 2.5-fold (p less than 0.001) and reduced the rate of phosphatidylcholine biosynthesis by approximately 40%. Aminophylline, 8-bromoadenosine 3':5'-monophosphate and N6,O2'-dibutyryladenosine 3':5'-monophosphate also inhibited [methyl-3H]choline incorporation into phosphatidylcholine. Although choline kinase and phosphocholinetransferase activities were stimulated by chlorophenylthio-cAMP treatment, CTP: phosphocholine cytidylyltransferase activity was reduced 46% (p less than 0.01). The results indicate that cytidylyltransferase may be phosphorylated and inhibited by cAMP-dependent protein kinases.     

Effects of ursodeoxycholic acid, analogues of ursodeoxycholic acid and combination of bile acids on bile acid synthesis in cultured rat hepatocytes

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

Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes

The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of [14C]cholesterol from [2-14C]acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of [14C]cholesterol from [2-14C]acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis.     

Feedback inhibition of bile acid synthesis in cultured pig hepatocytes

Bile acid synthesis by cultured pig hepatocytes, as measured by conversion of [14C]cholesterol to bile acids, increased during the second and third day of culture. This rise was inhibited after addition of various conjugated and unconjugated bile acids in a concentration of 100 microM. It could be completely prevented by cycloheximide, indicating that de novo protein synthesis is required for the increase in bile acid formation. No effect of exogenous bile salts on LDH release to the medium or on cellular ATP content was observed, demonstrating that hepatocyte viability was not affected. During the period in which bile acid synthesis was inhibited, pig hepatocytes were able to accumulate taurocholic acid (100 microM) up to 7-18 nmol per mg cell protein (decreasing during culture time). It is concluded that feedback regulation of bile acid synthesis is exerted by direct action of bile acids on the hepatocyte.     

Maintenance of bile acid synthesis and cholesterol 7 alpha-hydroxylase activity in cultured rat hepatocytes.

Addition of foetal-bovine serum to rat hepatocytes cultured in Williams E medium resulted in improved maintenance of bile-acid-synthetic capacity and cholesterol 7 alpha-hydroxylase activity as compared with cultures supplemented with rat or newborn-bovine serum or cultures in a hormonally defined serum-free medium. Minimally, 5% (v/v) foetal-bovine serum was necessary to maintain these liver-specific functions. Serum factor(s) responsible for these effects were not dialysable or associated with lipoproteins, but were removed by charcoal extraction.     

Regulation of bile acid synthesis in cultured rat hepatocytes: stimulation by apoE-rich high density lipoproteins

Cultured rat hepatocytes obtained by liver perfusion with collagenase in the presence of soybean trypsin inhibitor were used to examine the role of high density lipoproteins (HDL) in supplying cholesterol to the hepatocyte for bile acid synthesis. Within 6 hr of adding HDL (d 1.07-1.21 g/ml) obtained from rat serum there was a significant stimulation of bile acid synthesis and secretion that reached 2-fold after 24 hr. The stimulation by HDL occurred at normal plasma concentrations (i.e., 500 micrograms/ml) and showed further stimulation in a dose-dependent manner reaching a maximum stimulation of 2- to 2.5-fold. The stimulation of bile acid synthesis was dependent on the cholesteryl ester content of the HDL. Several lines of evidence show that the HDL is taken up by a receptor-mediated process dependent on apoE. These include: 1) at the same concentration (500 micrograms/ml) apoE-poor HDL (not retained by heparin affinity chromatography of HDL isolated from the plasma of rats fasted for 72 hr stimulated bile acid synthesis by 48%, whereas apoE-rich HDL stimulated bile acid synthesis by 110%; 2) reductive methylation totally blocked the stimulation of bile acid synthesis by HDL; 3) HDLC, which contained apoE as its major protein component, also maximally stimulated bile acid synthesis; and 4) human HDL, which contained no detectable apoE, failed to stimulate bile acid synthesis. Additional studies showed that apoE-enriched HDL and HDLC both inhibited cholesterol synthesis (determined by the incorporation of 3H2O) and caused a net accumulation of cholesteryl esters in hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Choline metabolism and phosphatidylcholine biosynthesis in cultured rat hepatocytes.

1. Adult rat hepatocytes were isolated by collagenase perfusion and were maintained in monolayer culture for 24h. 2. Choline metabolism and phosphatidylcholine biosynthesis were studied in these cells by performing pulse-chase studies at physiological concentrations (1-40 microM) of (Me-3H)-labelled or unlabelled choline in the culture medium. 3. During the 15 min pulse incubation, choline entering the cells was rapidly phosphorylated to phosphocholine or oxidized to betaine. Low concentrations of choline in the medium decreased the relative amount of choline oxidized. 4. During the 3 h chase period, the radioactivity in the phosphocholine pool was transferred to phosphatidylcholine. Very little radioactivity was associated with CDP-choline. These results provide good evidence that the rate-limiting step for phosphatidylcholine biosynthesis in these cultured hepatocytes is the conversion of phosphocholine into CDP-choline. Similar results were obtained for all concentrations of choline in the culture medium. 5. Cellular concentrations of phosphocholine were unaffected by the concentration of choline (1-40 microM) in the medium. 6. The majority of the label associated with betaine was secreted into the culture medium during the chase incubation. 7. From the pulse-chase studies, and the cellular phosphocholine concentrations, it was possible to estimate the rate of phosphatidylcholine biosynthesis (2.2, 2.8, 3.1 and 3.7 nmol/min per g wet weight of cells cultured in 1, 5, 10 and 40 microM-choline respectively for up to 4.25 h).     

Inhibition of dolichyl phosphate biosynthesis by compactin in cultured rat hepatocytes

Isolated rat hepatocytes were cultured in monolayer for about 24 h. During this period the cells exhibited constant protein and lipid synthesis. When the culture medium contained compactin, a competitive inhibitor of the 3-hydroxyl-3-methylglutary-coenzyme-A reductase, dolichyl-P synthesis was inhibited by 91% at the end of the incubation, as estimated by the incorporation of [3H]acetate and by 77% as estimated by the incorporation of 32Pi. These results indicate that in primary cultures of rat hepatocytes dolichyl monophosphate is mainly synthesized through a de novo process, while phosphorylation through the CTP-mediated kinase is of limited functional importance.     

Redundant pathways for negative feedback regulation of bile acid production

The orphan nuclear hormone receptor SHP has been proposed to have a key role in the negative feedback regulation of bile acid production. Consistent with this, mice lacking the SHP gene exhibit mild defects in bile acid homeostasis and fail to repress cholesterol 7-alpha-hydroxylase expression in response to a specific agonist for the bile acid receptor FXR. However, this repression is retained in SHP null mice fed bile acids, demonstrating the existence of compensatory repression pathways of bile acid signaling. We provide evidence for two such pathways, based on activation of the xenobiotic receptor PXR or the c-Jun N-terminal kinase JNK. We conclude that redundant mechanisms regulate this critical aspect of cholesterol homeostasis.     

Okadaic acid inhibits phosphatidylethanolamine biosynthesis in rat hepatocytes.

Okadaic acid, a specific inhibitor of protein phosphatase 1 and 2A, inhibited the synthesis of phosphatidylethanolamine via the CDPethanolamine pathway in isolated hepatocytes. Pulse-chase experiments and measurement of the enzyme activity demonstrated that the inhibition of phosphatidylethanolamine synthesis was not caused by an inhibition of CTP:phosphoethanolamine cytidylyltransferase, the putative regulatory enzyme. However, okadaic acid decreased the cellular diacylglycerol level to 30% of that in control cells. The data suggest that the availability of diacylglycerol limits phosphatidylethanolamine synthesis in okadaic acid-treated hepatocytes.     

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.     

Exogenous myristic acid acylates proteins in cultured rat hepatocytes

Fatty acid acylation is a functionally important modification of proteins. In the liver, however, acylated proteins remain largely unknown. This work was aimed at investigating fatty acid acylation of proteins in cultured rat hepatocytes. Incubation of these cells with [9,10-3H] myristic acid followed by two-dimensional electrophoresis separation of the delipidated cellular proteins and autoradiography evidenced the reproducible and selective incorporation of radioactivity from the precursor into 18 well-resolved proteins in the 10--120 kDa range and the 4--7 pH range. Radiolabeling of these proteins resulted from covalent linkage to the precursor [9,10-3H] myristic acid or to its elongation product, palmitic acid. The majority of the covalent linkages between the proteins and the fatty acids were broken by base hydrolysis, which indicated that the linkage was of thioester or ester-type. Only one of the studied proteins was attached to myristic acid via an amide linkage which resisted the basic treatment but was broken by acid hydrolysis. After incubation with [9,10-3H] palmitic acid, only two proteins previously detected with myristic acid were radiolabeled. Finally, the identified acylated proteins may be grouped into two classes: proteins involved in signal transduction (the alpha subunit of a heterotrimeric G protein and several small G proteins) and cytoskeletal proteins (cytokeratins, actin).     

Regulation of sterol 27-hydroxylase and an alternative pathway of bile acid biosynthesis in primary cultures of rat hepatocytes

In man, hepatic mitochondrial sterol 27-hydroxylase and microsomal cholesterol 7-hydroxylase initiate distinct pathways of bile acid biosynthesis from cholesterol, the “acidic” and “neutral” pathways, respectively. A similar acidic pathway in the rat has been hypothesized, but its quantitative importance and ability to be regulated at the level of sterol 27-hydroxylase are uncertain. In this study, we explored the molecular regulation of sterol 27-hydroxylase and the acidic pathway of bile acid biosynthesis in primary cultures of adult rat hepatocytes. mRNA and protein turnover rates were approximately 10-fold slower for sterol 27-hydroxylase than for cholesterol 7-hydroxylase. Sterol 27-hydroxylase mRNA was not spontaneously expressed in culture. The sole requirement for preserving sterol 27-hydroxylase mRNA at the level of freshly isolated hepatocytes (0 h) after 72 h was the addition of dexamethasone (0.1 μM; > 7-fold induction). Sterol 27-hydroxylase mRNA, mass and specific activity were not affected by thyroxine (1.0 μM), dibutyryl-cAMP (50 μM), nor squalestatin 1 (150 nM-1.0 μM), an inhibitor of cholesterol biosynthesis. Taurocholate (50 μM), however, repressed sterol 27-hydroxylase mRNA levels by 55%. Sterol 27-hydroxylase specific activity in isolated mitochondria was increased > 10-fold by the addition of 2-hydroxypropyl-β-cyclodextrin. Under culture conditions designed to maximally repress cholesterol 7-hydroxylase and bile acid synthesis from the neutral pathway but maintain sterol 27-hydroxylase mRNA and activity near 0 h levels, bile acid synthesis from [¹⁴C]cholesterol remained relatively high and consisted of β-muricholate, the product of chenodeoxycholate in the rat. We conclude that rat liver harbors a quantitatively important alternative pathway of bile acid biosynthesis and that its initiating enzyme, sterol 27-hydroxylase, may be slowly regulated by glucocorticoids and bile acids.     

Absence of bicarbonate abolishes the glycogenic effect of cortisol in cultured fetal rat hepatocytes

The glycogenic action of cortisol in cultured fetal rat hepatocytes was completely abolished by the absence of NaHCO3 from the medium, while its presence stimulated the action in relation to its concentration. The absence of NaHCO3 slightly reduced glycogen storage by insulin but did not affect glucose-dependent glycogen deposition in the basal state. Also, the cortisol-induced increase in glycogen synthase a activity was reduced but that in total synthase activity was not affected. The absence of NaHCO3 did not reduce the cortisol-induced increase in tyrosine aminotransferase activity and the incorporation of [3H]dexamethasone into the nuclei. These results show that the absence of NaHCO3 specifically inhibits the glycogenic action of glucocorticoids in cultured fetal rat hepatocytes and indicate the need for further investigation into the role of HCO3- in universally used bicarbonate-buffered media.     

Autoregulatory control of actin synthesis in cultured rat hepatocytes.

ADP-ribosylation of actin by Clostridium botulinum C2 toxin resulted in a depolymerization of filamentous F-actin and an increase of monomeric G-actin in cultured hepatocytes. Simultaneously the de novo synthesis of actin was largely reduced, while the synthesis of albumin and of other proteins was not significantly impaired. The specific decrease of actin mRNA to 30% of the control indicates a down-regulation of actin synthesis at a pretranslational level. On the other hand, treatment with the mycotoxin phalloidin resulted in an increase of F-actin and a decrease of monomeric G-actin. Under this condition the de novo synthesis of actin was specifically enhanced and the level of actin mRNA was increased to 600% of the control. The data suggest an autoregulatory control of the actin synthesis.     

Cysteine uptake and taurine biosynthesis in freshly isolated and primary cultured rat hepatocytes

Analysis of the uptake and metabolism of [14C]cysteine in rat liver was undertaken using freshly isolated hepatocytes and hepatocytes maintained in primary culture. The uptake of [14C]cysteine by freshly isolated hepatocytes was by means of both saturable and non-saturable transport systems and the former system was thought to involve facilitated diffusion. The uptake of [14C]cysteine by hepatocytes maintained in primary culture for 24 h also consisted of non-saturated and saturated transport mechanisms. The magnitude of the saturable transport system in cultured hepatocytes was, however, much greater than that found in freshly isolated hepatocytes, and was considered to be operated by active transport. Both freshly isolated and primary cultured hepatocytes had cysteine sulphinic acid decarboxylase activity, but this enzyme activity in the latter cells was noticeably reduced in comparison with that found in freshly isolated hepatocytes. Hepatocytes maintained in primary culture produced not only radiolabelled taurine, but also radiolabelled cysteine sulphinic acid, hypotaurine and alanine when incubated with [14C]cysteine. The present results indicate that cultured hepatocytes actively transport cysteine as well as metabolizing cysteine to taurine via cysteine sulphinic acid and hypotaurine.