Retinol and retinyl esters in parenchymal and nonparenchymal rat liver cell fractions after long-term administration of ethanol

Chronic ethanol consumption reduces the liver retinoid store in man and rat. We have studied the effect of ethanol on some aspects of retinoid metabolism in parenchymal and nonparenchymal liver cells. Rats fed 36% of total energy intake as ethanol for 5-6 weeks had the liver retinoid concentration reduced to about one-third, as compared to pair-fed controls. The reduction in liver retinoid affected both the parenchymal and the nonparenchymal cell fractions. Plasma retinol level was normal. Liver uptake of injected chylomicron [3H]retinyl ester was similar in the experimental and control group. The transport of retinoid from the parenchymal to the nonparenchymal cells was not found to be significantly retarded in the ethanol-fed rats. Despite the reduction in total retinoid level in liver, the concentrations of unesterified retinol and retinyl oleate were increased in the ethanol fed rats. Hepatic retinol esterification was not significantly affected in the ethanol-fed rats. Since our study has demonstrated that liver uptake of chylomicron retinyl ester is not impaired in the ethanol-fed rat, we suggest that liver retinoid metabolism may be increased.     

Lipoprotein lipase expression level influences tissue clearance of chylomicron retinyl ester

Approximately 25% of postprandial retinoid is cleared from the circulation by extrahepatic tissues. Little is known about physiologic factors important to this uptake. We hypothesized that lipoprotein lipase (LpL) contributes to extrahepatic clearance of chylomicron vitamin A. To investigate this, [3H]retinyl ester-containing rat mesenteric chylomicrons were injected intravenously into induced mutant mice and nutritionally manipulated rats. The tissue sites of uptake of 3H label by wild type mice and LpL-null mice overexpressing human LpL in muscle indicate that LpL expression does influence accumulation of chylomicron retinoid. Skeletal muscle from mice overexpressing human LpL accumulated 1.7- to 2.4-fold more 3H label than wild type. Moreover, heart tissue from mice overexpresssing human LpL, but lacking mouse LpL, accumulated less than half of the 3H-label taken up by wild type heart. Fasting and heparin injection, two factors that increase LpL activity in skeletal muscle, increased uptake of chylomicron [3H] retinoid by rat skeletal muscle. Using [3H]retinyl palmitate and its non-hydrolyzable analog retinyl [14C]hexadecyl ether incorporated into Intralipid emulsions, the importance of retinyl ester hydrolysis in this process was assessed. We observed that 3H label was taken up to a greater extent than 14C label by rat skeletal muscle, suggesting that retinoid uptake requires hydrolysis.In summary, for each of our experiments, the level of lipoprotein lipase expression in skeletal muscle, heart, and/or adipose tissue influenced the amount of [3H]retinoid taken up from chylomicrons and/or their remnants.     

Uptake and cellular transport of [11-3H] all-trans-retinoic acid in the liver of vitamin A-deficient hamsters

In this study, we examined, by ultrastructural autoradiography, the uptake and intracellular transport of [3H]all-trans-retinoic acid ([3H]RA) in the livers of vitamin A-deficient hamsters. Four-week-old animals were administered 25 microCi of [3H]RA by gavage, and, at different intervals thereafter, one animal was sacrificed. Their livers were excised and processed for autoradiography. Radioactive grains were observed to pass randomly through the plasma membrane by diffusion. No evidence of retinoid internalization by endocytosis was observed. Between 1 and 30 min after gavage, the radioactivity in parenchymal cells was associated mainly with rough endoplasmic reticulum (RER) and mitochondria. The labeling over nuclei was apparent at 1 min, remained relatively high up to 30 min, and subsequently decreased. At 2 and 5 hr, only a few grains were observed over nuclei, RER and mitochondria. At 24 hr, most of the labeling was associated with endothelial cells and sinusoidal spaces, indicating mobilization of [3H]RA from the liver. The results indicate that [3H]RA is transported through the plasma membrane by transmembrane diffusion without endocytosis and, after entering the cells, the ligand is rapidly translocated into nuclei.     

Chylomicron remnant-vitamin A metabolism by the human hepatoma cell line HepG2

The binding and metabolism of [3H]vitamin A-containing chylomicron (CM) remnants by the human hepatoma cell line HepG2 were studied. Mesenteric lymph chylomicrons were collected from [3H]retinol-fed rats and incubated with lipoprotein lipase to obtain CM remnants. At 4 degrees C, specific CM remnant binding was inhibited by an excess of unlabeled CM remnants. Specific binding predominated at low concentrations and approached saturation while total binding continued to increase over an extensive concentration range (0.45-32 microgram triglyceride/ml). CM remnant uptake at 37 degrees C was greater than that of CM and at least 70 times more efficient than the pinocytosis of sucrose. CM remnant binding increased with the extent of lipolysis. Addition of human apolipoprotein E enhanced both CM remnant and CM binding. After internalization, HepG2 cells hydrolyzed CM remnant-[3H]retinyl esters, and radiolabeled metabolites accumulated. As a function of the concentration of [3H]retinoid initially bound to cells, retinol and retinyl esters accumulated as the major cell-associated metabolites. In contrast, retinol was the major metabolite in the medium only at low retinoid concentrations; other more polar metabolites accumulated at higher concentrations (greater than 110 pmol retinoid/mg cell protein). The accumulation in the medium of labeled metabolites derived from CM remnant-retinoid was reduced when cells were preincubated in unlabeled retinol-supplemented media. The specific activity of retinol in the medium indicated that CM remnant-vitamin A had mixed with the cellular store prior to its secretion as retinol. These results indicate that HepG2 cells internalize CM remnants in part by specific binding sites, and that the metabolism of CM remnant-retinoids by the HepG2 cell involves retinyl ester hydrolysis and the secretion of retinol and other more polar metabolites. These processes were regulated in part by the concentration of retinoid delivered by the CM remnant and by the initial retinoid content of the cell.     

Uptake of vitamin A in macrophages from physiologic transport proteins: role of retinol-binding protein and chylomicron remnants

Vitamin A plays an important role in reducing infectious disease morbidity and mortality by enhancing immunity, an effect that is partly mediated by macrophages. Thus, knowing how these cells take up vitamin A is important. The results in the present study demonstrate that J774 macrophages efficiently take up chylomicron remnant retinyl esters and retinol-binding protein (retinol-RBP) bound retinol by specific and saturable mechanisms. The binding of (125)I-RBP to plasma membrane vesicles demonstrated that the macrophage receptor had a similar binding affinity, as was discovered previously for other cells. The B(max) for the macrophages was smaller than the values reported for placenta, bone marrow, and kidney, but larger than that reported for liver. The J774 cells also bound and took up [(3)H]retinol-RBP. Approximately 50 to 60% of the uptake may compete with excess unlabeled retinol-RBP and approximately 30 to 40% with excess transtyrethin. Following the uptake of [(3)H]retinol-RBP, an extensive esterification occurred: After 5 hours of incubation, 77.8 +/- 3.9% (SD; n = 3) of the cellular radioactivity was recovered as retinyl esters. The J774 cells also demonstrated saturable binding of chylomicron remnant [(3)H]retinyl esters, and a continuous uptake at 37 degrees C followed by an extensive hydrolysis of the retinyl esters. Binding could be inhibited by approximately 50% by excess unlabeled low density lipoprotein (LDL). In addition, lipoprotein lipase increased the binding of chylomicron remnant [(3)H]retinyl esters by approximately 30% and the uptake of chylomicron remnant [(3)H]retinyl ester by more than 300%. Furthermore, because sodium chlorate reduced binding with 40% and uptake with 55%, the results suggest that proteoglycans are involved in the uptake. Thus, the results suggest that both LDL receptor and LDL-related protein are involved in the uptake of chylomicron remnant [(3)H]retinyl ester in macrophages.     

Intracellular transport and degradation of chylomicron remnants in rat liver cells after in vivo endocytosis

The intracellular transport and degradation of in vivo endocytosed chylomicron remnants labelled with 125I in the protein moiety was studied in rat liver cells by means of subcellular fractionation in Nycodenz and sucrose density gradients. Initially, the radioactivity was located in low-density endosomes and was sequentially transferred to light and dense lysosomes. Data from gel filtration of the light and dense lysosomal fractions showed radioactive material with a molecular weight of about 1000-2000, representing short peptide fragments or amino acids which remain attached to iodinated tyramine cellobiose. In addition, undegraded apoproteins accumulated in both types of lysosome. Our data suggest that endocytosed chylomicron remnant apoproteins are first located in low-density endosomes and are sequentially transferred to light and dense lysosomes. Furthermore, the degradation process starts in the light lysosomes.     

Retinoylation of proteins in a macrophage tumor cell line J774, following uptake of chylomicron remnant retinyl ester

Following uptake of chylomicron remnant retinyl esters by the macrophage cell line J774, the retinyl esters are hydrolyzed to retinol before retinol is further metabolized to retinal and the various retinoic acid isoforms. One hour after the addition of chylomicron remnant [³H]retinyl esters to the cells, the percentage of cell-associated radioactivity in the retinyl ester fraction had decreased from approximately 90% to approximately 40%, whereas the radioactivity in the retinol fraction increased correspondingly. After 4 hours of incubation, more than 79% of the radioactive retinyl esters had been hydrolyzed to retinol. When we measured incorporation of radioactivity in the protein fraction, we observed that the level of [³H]retinoylated proteins increased rapidly the first 4 hours, and then continued to increase at a lower rate up to 24 hours, when approximately 0.6% of the cell-associated radioactivity was covalently bound to protein. These data suggest that approximately 0.18% of all the cellular proteins might be retinoylated under such conditions. In summary, in the present study we have demonstrated that retinoids taken up by a macrophage cell line as chylomicron remnant retinyl esters, a physiologic plasma transport molecule for vitamin A, might be covalently linked to proteins. Such retinoylation might be relevant both for normal function, as well as for the toxic and teratogenic effects of vitamin A.     

Hepatic processing and biliary secretion of the cholesteryl esters from beta very-low-density lipoproteins in the rat

beta-Migrating very-low-density lipoproteins (beta-VLDL) are cholesteryl-ester-enriched lipoproteins which accumulate in the serum of cholesterol-fed animals or patients with type III hyperlipoproteinemia. In the rat, beta-VLDL are rapidly cleared by the liver and parenchymal liver cells form the major site for uptake. In this investigation, beta-VLDL were labeled with [3H]cholesteryl esters and the hepatic intracellular transport of these esters was followed. 2 min after injection, the major part of the [3H]cholesteryl esters is already associated with the liver and a significant proportion is recovered in endosomes. Up to 25 min after injection, an increase in radioactivity in the lysosomal compartment is noticed. This radioactivity initially represents cholesteryl esters, while from 25 min onward, radioactivity is mainly present in unesterified cholesterol. Between 45 min and 90 min after beta-VLDL injection, specific transfer of unesterified [3H]cholesterol to the endoplasmic reticulum is observed, while by 3 h the majority is located in this fraction. The appearance of radioactivity in the bile was rather slow as compared to the rapid initial uptake and processing, and up to 5 h after injection only 10% of the injected dose had reached the bile (mainly as bile acids). 72 h after injection, the amount of the injected radioactivity recovered in the bile had increased to 50%. Chloroquine treatment of the rats inhibited the hydrolysis of the cholesteryl esters and the appearance of radioactivity in the bile was retarded. It is concluded that beta-VLDL are rapidly processed by parenchymal liver cells and that the cholesteryl esters from beta-VLDL are hydrolyzed in the lysosomal compartment. Unesterified cholesterol remains associated with the endoplasmic reticulum for a prolonged time, although ultimately the majority will be secreted into the bile as bile acids. The effective operation of this pathway will prevent extrahepatic accumulation of cholesteryl esters from beta-VLDL, while the prolonged residence time of unesterified cholesterol in the endoplasmic reticulum might be important for regulation of low-density lipoprotein (LDL) receptors in liver and thus for LDL levels in the blood.     

Binding of [3H]ctyochalasin B and [3H]colchicine to isolated liver plasma membranes.

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([3H]cytochalasin B and [3H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [3H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB3H4. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insensitive to changes of pH or ionic strength. At 10(-6) M [3H]cytochalasin B, glucose of p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 A) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10(-5) M [3H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [3H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes. [3H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [3H]cytochalasin B binding and cytochalasin B stimulated 3H colchicine binding. [3H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Perisinusoidal fat-storing cells are the main vitamin A storage sites in rat liver

Highly purified sinusoidal (fat-storing, Kupffer and endothelial cells) and parenchymal cells were isolated to assess the cellular distribution of vitamin A in liver of adult vitamin A-sufficient rats. A modified simple procedure was developed for the purification of fat-storing cells from rat liver. This was achieved by a single centrifugation step in a two-layer density Nycodenz gradient. Endothelial and Kupffer cells were obtained from the same gradient and further purified by centrifugal elutriation. Reverse-phase HPLC analysis showed that fat-storing cells contained about 300-fold the amount of retinyl esters present in parenchymal cells on a mg cell protein basis. In fat-storing cells, the same retinyl esters, viz. retinyl palmitate, retinyl stearate and retinyl oleate, were present as in whole liver. It was also observed that, within 12 h after intravenous injection of chylomicron [3H]retinyl ester, most of the radioactivity had accumulated in the fat-storing cells. It is concluded that fat-storing cells are the main storage sites for vitamin A in rat liver.     

Newly administered [3H]retinol is transferred from hepatocytes to stellate cells in liver for storage

We have recently shown that newly administered vitamin A (retinol) is initially taken up by the parenchymal cells of the liver, and subsequently (within 1-2 h) transferred to non-parenchymal liver cells (NPC) (Blomhoff et al., ref. [10]). In the present study we have separated the NPC by different methods to determine the cell type responsible for this uptake of [3H]retinol. When liver cells were prepared between 5 and 18 h after intraduodenal administration of [3H]retinol, the radioactive retinol was recovered mainly in the stellate cells. Other liver cells (i.e., hepatocytes, endothelial cells and Kupffer cells) contained only small amounts of [3H]retinol. Further, fluorescence microscopy studies indicated that stellate cells contain large quantities of retinol. Our results show that newly administered [3H]retinol, which is initially located in the hepatocytes, is transferred to the stellate cells and stored there.     

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.     

Lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine. Evidence for different pathways

The lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine has been studied. When rats were cannulated in the intestinal lymph duct and given an intraduodenal bolus of [3H]retinol and 14C-labelled vitamin D-3, 14C-labeled vitamin D-3 appeared later in the intestinal lymph than [3H]retinol and the rate of absorption of vitamin D-3 was still maximal at a time when that of retinol had declined. Both vitamins were absorbed via the lymphatic route in association with chylomicrons. Almost all the retinol was esterified, while vitamin D-3 appeared in the chylomicrons as free vitamin D-3. In vitro incubations and in vivo studies using hepatectomized and normal rats showed that the retinyl ester was a relatively nonexchangeable component of the chylomicrons and their remnants. Hence, all the vitamin A followed the remnants in their clearance from plasma. In contrast, significant amounts of vitamin D-3 were transferred from the chylomicrons to other plasma fractions. Therefore, only a fraction of this vitamin may be removed in association with the chylomicron remnants.     

Subcellular localization of the enzyme that forms mannosyl retinyl phosphate from guanosine diphosphate [14C]mannose and retinyl phosphate.

The subcellular distribution of the enzyme catalysing the conversion of retinyl phosphate and GDP-[14C]mannose into [14C]mannosyl retinyl phosphate was determined by using subcellular fractions of rat liver. Purity of fractions, as determined by marker enzymes, was 80% or better. The amount of mannosyl retinyl phosphate formed (pmol/min per mg of protein) for each fraction was: rough endoplasmic reticulum 0.48 +/- 0.09 (mean +/- S.D.); smooth membranes (consisting of 60% smooth endoplasmic reticulum and 40% Golgi apparatus), 0.18 +/- 0.03; Golgi apparatus, 0.13 +/- 0.03; and plasma membrane 0.02.     

Intracellular degradation of asialoglycoproteins in hepatocytes starts in a subgroup of lysosomes

Isolated rat hepatocytes take up and degrade [125I]tyramine-cellobiose-labelled asialofetuin [( 125I]TC-AF). The labelled degradation products are trapped at the site of degradation. The intracellular transport of [125I]TC-AF was studied by means of cell fractionation in Nycodenz gradients. The labelled ligand was kept in a small, slowly sedimenting vesicle during the first minutes after uptake in the cells, and was then transferred to a larger endosome. Labelled degradation products first appeared in an organelle with the same density distribution as the larger endosome and then in a denser organelle. These observations suggest that two types of lysosome, 'light' lysosomes and 'dense', are sequentially involved in the degradation of the asialoglycoprotein. The bulk of the lysosomal enzymes is associated with the dense lysosome.     

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.     

Intracellular transport of asialoglycoproteins in rat hepatocytes : Evidence for two subpopulations of lysosomes

The intracellular transport and degradation of asialoorosomucoid (AOM) in isolated rat hepatocytes was studied by means of subcellular fractionation in Nycodenz gradients. The asialoglycoprotein was labelled by covalent attachment of a radioiodinated tyramine-cellobiose adduct ( [125I]TC) which leads to labelled degradation products being trapped intracellularly and thus serving as markers for the degradative organelles. The ligand was initially (1 min) in a slowly sedimenting (small) vesicle and subsequently in larger endosomes. Acid-soluble, radioactive degradation products were first found in a relatively light lysosome whose distribution coincided in the gradient with that of the larger endosome. Later (30 min) degradation products were found in denser lysosomes which banded in the same region of the gradient as the lysosomal enzyme, beta-acetylglucosaminidase. Colchicine, monensin and leupeptin all inhibited degradation of [125I]tyramine-cellobiose asialoorosomucoid ( [125I]TC-AOM) and reduced the formation of degradation products in both the light and the dense lysosomes. In presence of monensin and colchicine no undegraded ligand was seen in the dense lysosome, suggesting that uptake in these vesicles was inhibited. Leupeptin allowed accumulation of undegraded ligand in the dense lysosome. Therefore, transfer from light to dense lysosomes is not dependent on degradation as such. In the presence of monensin two peaks of undegraded ligand were found in the gradients. It seems possible that in the monensin-sensitive endosomes, dissociation of the ligand-receptor complex is inhibited, allowing ligand to recycle with the receptors in small vesicles.     

The secretion of collagen by insects: uptake of [3H]proline by collagen-synthesizing cells in Locusta migratoria and Galleria mellonella.

The uptake of [H3]proline by collagen-secreting cells of the locust, Locusta migratoria, and wax-moth, Galleria mellonella, has been investigated by electron autoradiography. The locust cells are around the ejaculatory duct and they secrete collagen in the young adult male, while the wax-moth cells are those which produce the dorsal mass of connective tissue on the abdominal nerve cord during the late pupal stage. The cells were exposed to [H3]proline either by injection of the [3H]proline into the insect, or as a pulse while the tissue was maintained in a culture medium. The tissues were fixed at differeing experimental times after exposure to the [3H]proline. The resulting electron autoradiographs were subjected to quantitative analysis, and the silver grain distribution was determined as the relative number of grains per unit area over a series of tissue compartments. When the results of this analysis for the matrix, rough endoplasmic reticulum and Golgi complexes of the two tissues were plotted against experimental time, it was seen that the relative number of grains per unit area over the rough endoplasmic reticulum decreases while that over the matrix increases; statistical analysis has shown that these changes are significant. For the Golgi complexes, however, the theoretical variances are much greater, due to the small relative area occupied by this organelle. There is little evidence for anything other than random sampling fluctuations in the relative numbers of grains per unit area, and hence it is unlikely that the time course of the label over the Golgi complexes follows that over the rough endoplasmic reticulum. The conclusions drawn from these experiments are firstly that a large portion of the labelled protein passes straight from the rough endoplasmic reticulum to the matrix, but that a smaller portion of the labelled material might pass from the rough endoplasmic reticulum to the Golgi complexes and thence to the matrix. It is assumed that collagen comprises most of the protein which passes straight from the rough endoplasmic reticulum to the matrix, and while there is no evidence to exclude collagen from the material passing through the Golgi complexes, it is probable that other proteins and glycosaminoglycans are also present in this labelled material. These conclusions about the intracellular pathway for collagen secretion are similar to those derived from recent studies of some vertebrate fibroblasts. There is, however, conflicting opinion about the intracellular pathway of collagen and it is pointed out that there is diversity in collagen-synthesizing cells, which may account for the differences in the intracellular pathways for collagen secretion which have been proposed.     

Lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine Evidence for different pathways

The lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine has been studied. When rats were cannulated in the intestinal lymph duct and given an intraduodenal bolus of [³H]retinol and ¹⁴C-labelled vitamin D-3, ¹⁴C-labeled vitamin D-3 appeared later in the intestinal lymph than [³H]retinol and the rate of absorption of vitamin D-3 was still maximal at a time when that of retinol had declined. Both vitamins were absorbed via the lymphatic route in association with chylomicrons. Almost all the retinol was esterified, while vitamin D-3 appeared in the chylomicrons as free vitamin D-3. In vitro incubations and in vivo studies using hepatectomized and normal rats showed that the retinyl ester was a relatively nonexchangeable component of the chylomicrons and their remnants. Hence, all the vitamin A followed the remnants in their clearance from plasma. In contrast, significant amounts of vitamin D-3 were transferred from the chylomicrons to other plasma fractions. Therefore, only a fraction of this vitamin may be removed in association with the chylomicron remnants.     

Precursors of ricin and Ricinus communis agglutinin. Glycosylation and processing during synthesis and intracellular transport

During synthesis in vivo the castor bean lectin precursors initially appear in the endoplasmic reticulum as a group of core glycosylated polypeptides of relative molecular mass 64 000-68 000. Pretreatment of intact castor bean endosperm tissue with tunicamycin partially inhibits the cotranslational core glycosylation step and results in the accumulation of a single sized unglycosylated precursor polypeptide of relative molecular mass 59 000. The glycosylated precursors in the endoplasmic reticulum were enzymically converted to the 59 000-Mr form by incubation with endoglucosaminidase H. Intracellular transport of the glycosylated lectin precursors from the endoplasmic reticulum to a denser vesicle fraction was accompanied by modifications to the oligosaccharide moieties which conferred resistance to the action of endoglucosaminidase H. The post-translational addition of fucose to the carbohydrate chain was identified as one of the oligosaccharide modification steps. Fucose addition was catalysed by a glycosyltransferase associated with a smooth-surfaced membrane fraction which was distinct from the endoplasmic reticulum and which was tentatively identified as the Golgi apparatus. Glycosylation was not essential for intracellular transport of the lectin precursors: unglycosylated precursor synthesized in the presence of tunicamycin gave rise to unglycosylated lectin subunits in the protein bodies.