Interactions of Transthyretin (TTR) and Retinol-Binding Protein (RBP) in the Uptake of Retinol by Primary Rat Hepatocytes

The mechanism by which cells take up retinol from retinol-binding protein (RBP) and the role of the RBP–transthyretin (TTR) complex remain unclear. Here we report on retinol uptake through the RBP–TTR complex by primary cultured rat hepatocytes (parenchymal cells, PC) and nonparenchymal cells (NPC) following incubation with [³H]retinol–RBP or the [³H]retinol–RBP–TTR complex under several conditions. The cellular accumulation of retinol was time and temperature dependent in both PC and NPC. Analysis by HPLC showed that the incorporated [³H]retinol in NPC was mainly converted to retinyl ester, although in PC it remained mainly as unesterified retinol. However, the amount of retinol taken up from the RBP–TTR complex was nearly twofold greater than that from RBP alone. The uptake of [³H]retinol from protein-bound retinol was inhibited by an excess of either retinol–RBP or retinol–RBP–TTR complex. Moreover, retinol uptake through the RBP–TTR complex was inhibited by an excess of free TTR. From these results we postulate that TTR may take part as a positive regulator in the delivery of RBP-bound retinol from plasma, possibly by a membrane receptor, and that retinol uptake takes place preferentially from the RBP–TTR complex into both PC and NPC. The uptake of [³H]retinol (2 μM) by PC was saturated, whereas uptake by NPC was not. These results indicate that the physiological importance of TTR in retinol delivery may be especially important to vitamin A-storing stellate (Ito) cells in the NPC fraction.     

Direct mobilization of retinol from hepatic perisinusoidal stellate cells to plasma.

We have studied the mechanism for mobilization of retinol from stellate cells. Our data show that perisinusoidal stellate cells isolated from liver contained retinol-binding protein (RBP) mRNA. By Western blot analysis we found that cultivated liver stellate cells secreted RBP into the medium. Cultivated stellate cells loaded in vitro with [3H]retinyl ester mobilized radioactive retinol as a complex with RBP. Furthermore, exogenous RBP added to the medium of cultured stellate cells increased the secretion of retinol to the medium. These data suggest that liver stellate cells in vivo mobilize retinol directly to the blood and that a transfer to parenchymal cells for secretion as holo-RBP is not required. The direct mobilization of retinol from liver stellate cells as retinol-RBP to blood is indirectly supported by the demonstration of RBP mRNA production and RBP secretion by lung stellate cells. The data suggest that the same mechanism for retinol mobilization may exist in hepatic and extrahepatic stellate cells. This is, vitamin A-storing stellate cells in liver, lungs, and probably also in other organs may synthesize their own RBP (or alternatively use exogenous RBP) and mobilize holo-RBP directly to the blood.     

Hepatic uptake of [3H]retinol bound to the serum retinol binding protein involves both parenchymal and perisinusoidal stellate cells

We have studied the hepatic uptake of retinol bound to the circulating retinol binding protein-transthyretin complex. Labeled complex was obtained from the plasma of donor rats that were fed radioactive retinol. When labeled retinol-retinol binding protein-transthyretin complex was injected intravenously into control rats, about 45% of the administered dose was recovered in liver after 56 h. Parenchymal liver cells were responsible for an initial rapid uptake. Perisinusoidal stellate cells initially accumulated radioactivity more slowly than did the parenchymal cells, but after 16 h, these cells contained more radioactivity than the parenchymal cells. After 56 h, about 70% of the radioactivity recovered in liver was present in stellate cells. For the first 2 h after injection, most of the radioactivity in parenchymal cells was recovered as unesterified retinol. The radioactivity in the retinyl ester fraction increased after a lag period of about 2 h, and after 5 h more than 60% of the radioactivity was recovered as retinyl esters. In stellate cells, radioactivity was mostly present as retinyl esters at all time points examined. Uptake of retinol in both parenchymal cells and stellate cells was reduced considerably in vitamin A-deficient rats. Less than 5% of the injected dose of radioactivity was found in liver after 5-6 h (as compared to 25% in control rats), and the radioactivity recovered in liver from these animals was mostly in the unesterified retinol fraction. Studies with separated cells in vitro suggested that both parenchymal and stellate cells isolated from control rats were able to take up retinol from the retinol-retinol binding protein-transthyretin complex. This uptake was temperature dependent.     

Retinol esterification in cultured rat liver cells.

Retinol esterification was examined in cultured hepatocytes and stellate cells from the rat. Esterification of [3H]retinol was linear for 2 h in both cell types. By increasing the concentration of retinol in the medium, there was a marked increase in retinol esterification in both cell types. The capacity for esterification of retinol was in the same order of magnitude in the two cell types at 3.5 microM-retinol in the medium. This represents a rate of retinol esterification which far exceeds that required to esterify the amount of retinol absorbed in the intestine. It was demonstrated in particulate homogenates from cultured hepatocytes that the esterification of retinol was dependent on acyl-CoA. Addition of 25-hydroxycholesterol or mevalonolactone promoted an increase in cholesterol esterification, whereas retinol esterification was unaffected, suggesting that cholesterol and retinol are esterified by two different enzymes. Some 80% of vitamin A in cultured hepatocytes is retinyl esters, mostly retinyl palmitate. By adding 87 microM-retinol in the medium the cells accumulated 100-fold free retinol and 2.5-3.0-fold retinyl esters within 1 h. When retinol-loaded cells were incubated without retinol, there was a marked decrease especially in free but also in esterified retinol. In the presence of 1 mM-oleic acid in the medium the amount of retinyl oleate was twice that in control cells.     

Uptake and metabolism of retinol in cultured Sertoli cells: evidence for a kinetic model

When cultured Sertoli cells derived from 20-day-old weanling rats were supplied [3H]retinol bound to serum retinol binding protein-transthyretin complex, [3H]retinol was rapidly incorporated and [3H]retinyl esters were synthesized. Within 28 h after administration, 83% of the labeled retinoids were accounted for as retinyl esters (64% as retinyl palmitate). Sertoli cells derived from vitamin A deficient rats and supplied [3H]retinol in culture under identical conditions likewise incorporated [3H]retinol and synthesized retinyl esters. In contrast to normal Sertoli cells, vitamin A deficient Sertoli cells eventually metabolized virtually all of the cellular [3H]retinol to retinyl esters. The primary metabolic fate of retinol administered to Sertoli cell cultures was the synthesis of retinyl esters under all conditions tested. However, administration of [3H]retinol bound to serum retinol binding protein gave metabolic profiles having a higher proportion of retinyl esters and lower proportions of unresolved polar material than administration of [3H]retinol alone. The kinetics of retinol uptake and intracellular retinyl ester synthesis in cultured Sertoli cells was complex. An initial, rapid phase of [3H]retinol incorporation lasting 30 min was followed by a slower rate of incorporation and a concomitant decrease in the intracellular concentration of [3H]retinol. During the time course the specific activity of [3H]retinyl palmitate eventually exceeded that of intracellular [3H]retinol. These observations suggest that two intracellular pools of retinol may exist in Sertoli cells.     

Metabolism of HDL-cholesteryl ester in the rat, studied with a nonhydrolyzable analog, cholesteryl linoleyl ether

Intralipid was sonicated with [3H]cholesteryl linoleyl ether (a nonhydrolyzable analog of cholesteryl linoleate) and incubated with rat HDL and d greater than 1.21 fraction of rabbit serum at a ratio of 0.012 mg triacylglycerol to 1 mg HDL protein. 25% of [3H]cholesteryl linoleyl ether was transferred to HDL. The labeled HDL was injected into donor rats and was screened for 4 h. [125I]HDL was subjected to the same protocol as the 3H-labeled HDL, including screening. The screened, labeled sera were injected into acceptor rats and the disappearance of radioactivity from the circulation was compared. The t1/2 in the circulation of [125I]HDL was about 10.5 h, while that of [3H]cholesteryl linoleyl ether-HDL was about 8 h. The liver and carcass were the major sites of uptake of [3H]cholesteryl linoleyl ether-HDL and accounted for 29-41% (liver) and 30% (carcass) of the injected label. Maximal recovery of [3H]cholesteryl linoleyl ether in the liver was seen 48 h after injection, and thereafter there was a progressive decline of radioactivity, which reached 7.8% after 28 days. The maximal recovery of [125I]HDL in the liver was about 9%. Pretreatment of the acceptor rats with estradiol for 5 days resulted in a 20% increase in the hepatic uptake of [3H]cholesteryl linoleyl ether-HDL and a 5-fold increase in adrenal uptake. The present findings indicate that in the rat the liver is the major site of uptake of HDL cholesteryl ester and that part of the HDL cholesteryl ester may be cleared from the circulation separately from the protein moiety. On the basis of our previous findings (Stein, Y., Kleinman Y, Halperin, G., and Stein, O. (1983) Biochim. Biophys. Acta 750, 300-305) the loss of the [3H]cholesteryl linoleyl ether from the liver after 14-28 days was interpreted to indicate that the labeled [3H]cholesteryl linoleyl ether had been taken up by hepatocytes.     

Expression of carboxylesterase and lipase genes in rat liver cell-types

Approximately 80% of the body vitamin A is stored in liver stellate cells with in the lipid droplets as retinyl esters. In low vitamin A status or after liver injury, stellate cells are depleted of the stored retinyl esters by their hydrolysis to retinol. However, the identity of retinyl ester hydrolase(s) expressed in stellate cells is unknown. The expression of carboxylesterase and lipase genes in purified liver cell-types was investigated by real-time PCR. We found that six carboxylesterase and hepatic lipase genes were expressed in hepatocytes. Adipose triglyceride lipase was expressed in Kupffer cells, stellate cells and endothelial cells. Lipoprotein lipase expression was detected in Kupffer cells and stellate cells. As a function of stellate cell activation, expression of adipose triglyceride lipase decreased by twofold and lipoprotein lipase increased by 32-fold suggesting that it may play a role in retinol ester hydrolysis during stellate cell activation.     

Intracellular transport of endocytosed chylomicron [3H]retinyl ester in rat liver parenchymal cells. Evidence for translocation of a [3H]retinoid from endosomes to endoplasmic reticulum

The intracellular transport of chylomicron remnants labeled with [3H]retinyl ester was studied in rat liver parenchymal cells by means of subcellular fractionation in Nycodenz and sucrose density gradients. The data presented indicate that endocytosed chylomicron remnant [3H]retinyl ester initially is located in low density endosomes. Radioactivity is subsequently transferred to a denser vesicle. Equilibrium as well as rate zonal centrifugation suggest that this denser [3H] retinoid-containing vesicle may represent endoplasmic reticulum. We have compared the intracellular transport of chylomicron remnant [3H]retinyl ester and 125I-asialofetuin. The receptor-mediated endocytosis of asialoglycoproteins in rat liver parenchymal cells is a thoroughly studied system. Our results suggest that the [3H] retinoid and 125I-asialofetuin follow the same path initially to the endosomes. After transit in endosomes, the intracellular transport differs. While asialofetuin is transported to the lysosomes, the retinoid is probably transferred to the endoplasmic reticulum.     

The stimulation by sodium fluoride of plasma-membrane Ca2+ inflow in isolated hepatocytes. Evidence that a GTP-binding regulatory protein is involved in the hormonal stimulation of Ca2+ inflow.

Bilirubin may be transported within intracellular membranes of the hepatocyte and may undergo membrane-membrane transfer to gain access to the conjugating enzyme UDP-glucuronyltransferase in the endoplasmic reticulum. We have demonstrated previously that the lipid composition of liposomal membranes incorporating bilirubin substrate influences the rate of transfer and glucuronidation of bilirubin by hepatic microsomes. To examine the mechanism(s) of substrate transfer, we incorporated radiolabelled bilirubin into small unilamellar model membranes of egg phosphatidylcholine or natural phospholipids in the proportions present in native hepatic microsomes. The rate at which bilirubin was transferred to rat liver microsomes and glucuronidated was then examined in the presence of various endogenous compounds that promote membrane fusion. For bilirubin substrate in membranes of egg phosphatidylcholine, the addition of Ca2+ (2 mM) increased the microsomal glucuronidation rate, whereas retinol enhanced microsomal conjugation rates for bilirubin in membranes of both lipid compositions. When the transfer of [3H]bilirubin from dual-labelled liposomes to microsomes was enhanced by Ca2+ or retinol, there was no associated increase in [14C]phospholipid transfer. Thus it appears likely that bilirubin is transferred to the endoplasmic reticulum by rapid cytosolic diffusion or membrane-membrane collisions, rather than by membrane fusion; this process may be modulated by changes in the lipid microenvironment of the substrate or the effective intracellular concentrations of Ca2+ or retinol. The observation that polymyxin B induced concomitant membrane-membrane transfer of [3H]bilirubin and [14C]phospholipid suggests that under certain circumstances membrane fusion or aggregation may promote the movement of lipophilic substrates in hepatocytes.     

Characteristics of retinol accumulation from serum retinol-binding protein by cultured Sertoli cells

The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoli cells accumulated [3H]retinol in a time- and temperature-dependent manner. At 32 degrees C, the rate of retinol accumulation was biphasic. Accumulation was linear for approximately 1 h, but then accumulation continued at a linear but decreased rate for 23 h. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/micrograms of cellular DNA. This amount of retinol was approximately equal to the cellular content of cellular retinol-binding protein (CRBP). Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with [3H]retinol-RBP for [3H]retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free [3H]retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 microM, suggesting that any change in the normal circulating retinol-RBP level (approximately 2 microM) would directly affect the rate of retinol accumulation. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. In addition, energy inhibitors and lysosomal poisons had no effect on [3H]retinol accumulation, indicating that RBP delivery of retinol to Sertoli cells did not occur by endocytosis of the retinol-RBP complex. Competition studies indicated, however, that protein recognition is important in the retinol uptake process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dolichol and retinol content of rat liver sinusoidal cells after chronic monensin treatment.

Aim of this study was to ascertain whether an impairment of communication between parenchymal and non-parenchymal liver cells involves vitamin A intercellular transport. The following approach was adopted: liver cells were isolated from rats treated chronically with the hydrophobic ionophore monensin i.p. for 3, 5, and 7 weeks and their retinol and dolichol content was assessed. Monensin, which alters membrane flow, was used because it had previously been reported to induce liver steatosis, cholestasis and glycogenolysis after acute treatment and, by preliminary morphological examination, to impair vitamin A transport between stellate cells and hepatocytes. Dolichol was chosen as a biochemical marker because it is a membrane lipid that modulates the fluidity and permeability of the membranes that retinol must cross. After monensin treatment, a load of vitamin A was given to rats three days before sacrifice, to ascertain whether its uptake by sinusoidal liver cells was altered. The main result was a dolichol decrease in hepatocytes and in the Ito-1 subfraction. In this latter, monensin induced a decrease in dolichol content only after vitamin A load. Moreover, while the hepatocytes were able to take up a load of vitamin A normally, the Ito-1 subfraction was no longer able to store retinol. Therefore the polarised transport of retinol between hepatocytes and stellate cells seemed impaired. The behaviour of sinusoidal endothelial cells and Kupffer cells might be ascribed to the functions of these cells and is not significantly modified by monensin. In conclusion, the altered cross-talk between sinusoidal cells in liver pathology might involve retinol as well as cytokines. Different pools of dolichol might have a role in this membrane process in a hydrophobic environment.     

Metabolism of [11-3H]retinyl acetate in liver tissues of vitamin A-sufficient, -deficient and retinoic acid-supplemented rats

A study was conducted on the incorporation of [11-3H]retinyl acetate into various retinyl esters in liver tissues of rats either vitamin A-sufficient, vitamin A-deficient or vitamin A-deficient and maintained on retinoic acid. Further, the metabolism of [11-3H]retinyl acetate to polar metabolites in liver tissues of these three groups of animals was investigated. Retinol metabolites were analyzed by high-performance liquid chromatography. In vitamin A-sufficient rat liver, the incorporation of radioactivity into retinyl palmitate and stearate was observed at 0.25 h after the injection of the label. The label was further detected in retinyl laurate, myristate, palmitoleate, linoleate, pentadecanoate and heptadecanoate 3 h after the injection. The specific radioactivities (dpm/nmol) of all retinyl esters increased with time. However, the rate of increase in the specific radioactivity of retinyl laurate was found to be significantly higher (66-fold) than that of retinyl palmitate 24 h after the injection of the label. 7 days after the injection of the label, the specific radioactivity between different retinyl esters were found to be similar, indicating that newly dosed labelled vitamin A had now mixed uniformly with the endogenous pool of vitamin A in the liver. The esterification of labelled retinol was not detected in liver tissues of vitamin A-deficient or retinoic acid-supplemented rats at any of the time point studied. Among the polar metabolites analyzed, the formation of [3H]retinoic acid from [3H]retinyl acetate was found only in vitamin A-deficient rat liver 24 h after the injection of the label. A new polar metabolite of retinol (RM) was detected in liver of the three groups of animals. The formation of 3H-labelled metabolite RM from [3H]retinyl acetate was not detected until 7 days after the injection of the label in the vitamin A-sufficient rat liver, suggesting that metabolite RM could be derived from a more stable pool of vitamin A.     

The use of newly synthesized phospholipids for assembly into secreted hepatic lipoproteins

We have recently shown (Vance, J.E. (1988) Biochim. Biophys. Acta 963, 70-81) that the percent distribution of molecular species of phosphatidylcholine (PC) derived from [methyl-3H]choline and [3-3H]serine, and phosphatidylethanolamine (PE) derived from [3-3H]serine were different in secreted lipoproteins and in the cultured hepatocytes from which the lipoproteins were produced. The species 1-stearoyl-2-arachidonoyl PC and PE were selectively not secreted. How this selection occurs is not known. One possible explanation is that secreted phospholipids are representative of the newly synthesized pool, whereas the molecular species composition of bulk cellular phospholipids has been altered by selective deacylation or by deacylation-reacylation. This hypothesis has been tested. The percent distribution of radioactivity from [1-3H]ethanolamine, [3-3H]serine and [methyl-3H]choline in nascent cellular and secreted PE and PC molecular species was examined by high-performance liquid chromatography. From [3H]serine labeling, the percent distribution of [3H]PE species in the medium after 4 h resembled closely that in cells 0.5 h, but not 4 h, after labeling. Thus, nascent phosphatidylserine-derived PE was immediately earmarked for secretion before remodeling occurred. Similarly, newly made rather than 'old' PE and PC from alternative biosynthetic sources may be preferred for assembly into lipoproteins. In addition, PE methyltransferase apparently preferred newly made, rather than remodelled, serine-derived PE for methylation to PC. In no instance (i.e., neither for any phospholipid nor any precursor) was there evidence that 'old' rather than 'new' phospholipid was specifically selected for secretion.     

Synthesis and metabolism of all-trans-[11-3H]retinyl beta-glucuronide in rats in vivo.

All-trans-[11-3H]retinyl beta-glucuronide (all-trans-[11-3H]ROG) was synthesized from [3H]retinol by an improved synthetic procedure. After its intraperitoneal injection into rats, ROG is initially found as the predominant labelled component in the serum, but then is distributed to the liver, intestine, kidney and other organs of the body. Esters of vitamin A, which constituted the major metabolite of ROG, were detected in the liver as well as in other tissues. Of the labelled vitamin A esters derived from tritiated ROG in the liver and intestine, about 50% contained 5,6-epoxyretinol, which was characterized by its chromatographic behaviour, formation of an acetyl ester and lack of reactivity with diazomethane. Thus ROG, although converted to retinol in vivo, might also act physiologically in an intact form.     

Pharmacokinetics,Tissue Distribution and Metabolism of Intravenously Administered Digalactosyldiacylglycerol and Monogalactosyldiacylglycerol in the Rat

Abstract

A bilayer forming galactolipid, digalactosyldiacylglycerol (DGalDG) has been identified as a tool with suitable physicochemichal properties for pharmaceutical formulation work. One possible application is as a carrier for liposome entrapped drugs for intravenous administration. The fate of intravenously administered galactolipids is not known. In this study liposomal dispersions of galactolipids, containing [³H]fatty acid labelled DGalDG or monogalactosyldiacylglycerol (MGalDG) were injected intravenously in the rat and the disappearance from blood and uptake by tissues were examined. The T1/2 of [³H]DGalDG in plasma was 3 to 5 minutes. Of the tissues examined (liver, spleen, kidneys, lung, heart, stomach, upper and lower small intestine and colon), the liver contained the highest radioactivity per g tissue after both 15 min. and 4 h. Autoradiographic examinations after 15 min, 1 h and 4 h showed that the uptake of radiolabeled DGalDG and MGalDG occurred mainly to the hepatocytes. Less than 6 % of the injected [³H]DGalDG remained in liver and plasma as [³H]DGalDG after 4 h. [³H]MGalDG exhibited a similar pattern of metabolism although the initial disappearance rate was faster than for [³H]DGalDG. The study thus shows that the hepatocytes take up and hydrolyse galactolipids after intravenous administration.     

Regulation of retinol uptake and esterification in MCF-7 and HepG2 cells by exogenous fatty acids.

The influence of extracellular fatty acids on the uptake and esterification of [3H]retinol bound to human retinol-binding protein (RBP), to RBP-transthyretin (TTR), or in dispersed form by the human hepatoma, HepG2, and human mammary epithelial carcinoma, MCF-7, cell lines was studied. The esterification of [3H]retinol was significantly increased in cells incubated with myristic, palmitic, stearic oleic, or linoleic acid-albumin complexes and was observed for all forms of [3H]retinol. Enhancement of [3H]retinol uptake was also observed in cells incubated with these fatty acids, but this increase was relatively small for the dispersed form as compared to that observed for [3H]retinol bound to RBP or RBP-TTR. Comparing equal concentrations of the [3H]retinol donors, cell uptake and esterification was greatest from the dispersed form and least from that bound to RBP-TTR. After preincubation of cells with oleate, uptake and esterification of [3H]retinol was increased but not to the extent observed when oleate and [3H]retinol donor were co-incubated. Incubation of cells with oleate resulted in rapid and correlated increases in the rates of [3H]retinol uptake and esterification which persisted until the steady state for [3H]retinol uptake was achieved. Beyond this time, net esterification of [3H]retinol continued in the presence of oleate. This kinetic pattern was observed for all [3H]retinol donors. These effects on [3H]retinol uptake and esterification were dose-dependent as the oleate to albumin ratio was varied from 0.5 to 3.0 and were observed across a physiological concentration range of RBP-3H-retinol. The data indicate that: 1) the fatty acid status of cells is a determinant of retinol uptake and esterification; and 2) the form of retinol presentation to cells is not qualitatively important for these processes.     

The uptake and metabolism of chylomicron-remnant lipids by rat liver parenchymal and non-parenchymal cells in vitro.

The uptake and metabolism of chylomicron-remnant lipids by individual liver cell types was examined by incubating remnants with monolayer cultures of hepatocytes, Kupffer cells, and endothelial cells from rat liver. Remnants were prepared in vitro from radiolabelled mesenteric-lymph chylomicra, utilizing either purified lipoprotein lipase from bovine milk, or plasma isolated from heparinized rats. The resulting particles contained [3H]phosphatidylcholine and cholesterol, and [14C]oleate in the acylglycerol, phospholipid, fatty-acid and cholesterol-ester fractions. The capacities of the three cell types for uptake of both [3H]lipids and [14C]lipids were determined to be, on a per-cell basis, in the order: Kupffer greater than hepatocytes greater than endothelial. The relative proportions of [3H]phospholipid and total [3H]cholesterol taken up by hepatocytes and non-parenchymal cells remained constant with time. The uptake of [14C]oleoyl lipids by all three cell types was slightly greater than that of the total [3H]cholesterol and [3H]phospholipid components. There was evidence of cholesterol-ester hydrolysis and turnover of [14C]oleate in the phospholipid fraction in hepatocytes and Kupffer cells, but not endothelial cells, over the first 2 h. With both remnant preparations, these observations indicate that significant differences exist between the three major liver cell types with respect to the uptake and metabolism of remnant lipid components.     

Acyl-CoA: retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT) activation during the lipocyte phenotype induction in hepatic stellate cells

We have examined retinol esterification in the established GRX cell line, representative of hepatic stellate cells, and in primary cultures of ex vivo purified murine hepatic stellate cells. The metabolism of [3H]retinol was compared in cells expressing the myofibroblast or the lipocyte phenotype, under the physiological retinol concentrations. Retinyl esters were the major metabolites, whose production was dependent upon both acyl-CoA:retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT). Lipocytes had a significantly higher esterification capacity than myofibroblasts. In order to distinguish the intrinsic enzyme activity from modulation of retinol uptake and CRBP-retinol content of the cytosol in the studied cells, we monitored enzyme kinetics in the purified microsomal fraction. We found that both LRAT and ARAT activities were induced during the conversion of myofibroblasts to lipocytes. LRAT induction was dependent upon retinoic acid, while that of ARAT was dependent upon the overall induction of the fat storing phenotype. The fatty acid composition of retinyl-esters suggested a preferential inclusion of exogenous fatty acids into retinyl esters. We conclude that both LRAT and ARAT participate in retinol esterification in hepatic stellate cells: LRAT's activity correlates with the vitamin A status, while ARAT depends upon the availability of fatty acyl-CoA and the overall lipid metabolism in hepatic stellate cells.     

Serum half-life, distribution, hepatic uptake and biliary excretion of alpha-tocopherol in rats

The serum clearance of alpha-[3H]tocopherol has been studied after intravenous injection of intestinal lymph labeled in vivo with radioactive alpha-tocopherol. The half-life of the injected alpha-[3H]tocopherol was approx. 12 min. Fractionation of plasma by ultracentrifugation 10 min after injection of lymph showed that 91% of the radioactive alpha-tocopherol remaining in plasma was located in chylomicrons (d less than 1.006 g/ml) and 7.8% in high-density lipoproteins (HDL, 1.05 less than d less than 1.21 g/ml). 2 h after administration of alpha-tocopherol, about 35% of the radioactivity recovered in plasma was associated with chylomicrons and approx. 51% with HDLs. alpha-[3H]Tocopherol was initially taken up by the liver, which contained more than 50% of the injected radioactivity after 45-60 min. Separation of parenchymal and nonparenchymal cells demonstrated a preferential uptake of alpha-[3H]tocopherol by the parenchymal liver cells. After 24 h about 11% of the injected dose was recovered in the liver. Considering whole organs the liver, adipose tissue and skeletal muscle had the highest content of radioactivity after 24 h. Furthermore, about 14% of the administered dose was recovered in bile during 24 h draining.     

Clearance of acetyl low density lipoprotein by rat liver endothelial cells. Implications for hepatic cholesterol metabolism

We have studied the hepatic uptake of human [14C] cholesteryl oleate labeled acetyl low density lipoprotein (LDL). Acetyl-LDL injected intravenously into rats was cleared from the blood with a half-life of about 10 min. About 80% of the injected acetyl-LDL was recovered in the liver after 1 h. Initially, most of the [14C]cholesterol was recovered in liver endothelial cells (about 60%). Some radioactivity (about 15%) was also recovered in the hepatocytes, while the Kupffer cells and stellate cells contained only small amounts of the label (less than 5%). About 1 h after injection, radioactivity started to disappear from endothelial cells and appeared instead in hepatocytes. Radioactivity subsequently declined in hepatocytes as well. After a lag phase of 4 h, significant amounts of radioactivity were recovered in bile. The in vitro uptake and hydrolysis of [14C]cholesteryl oleate-labeled acetyl-LDL were saturable in isolated rat liver endothelial cells. Native LDL does neither affect the uptake nor the hydrolysis of acetyl-LDL. Ammonia and monensin reduced the hydrolysis of acetyl-LDL in isolated liver endothelial cells. Furthermore, monensin at concentrations above 10 microM completely blocked the binding of acetyl-LDL to the liver endothelial cells, suggesting that the receptor for acetyl-LDL is trapped inside the cells. The liver endothelial cells may be involved in the protection against atherogenic lipoproteins, e.g. liver endothelial cells may mediate uptake of cholesterol from plasma and transfer of cholesterol to the hepatocytes for further secretion into the bile.