Retinoids, retinoid-binding proteins, and retinyl palmitate hydrolase distributions in different types of rat liver cells

A study was conducted to determine the levels and distributions of retinoids, retinol-binding protein (RBP), retinyl palmitate hydrolase (RPH), cellular retinol-binding protein (CRBP), and cellular retinoic acid-binding protein (CRABP) in different types of isolated liver cells. Highly purified fractions of parenchymal, fat-storing (stellate), endothelial, and Kupffer cells were isolated in high yield from rat livers. The retinoid content of each fraction was measured by HPLC analysis. RBP, CRBP, and CRABP were measured by sensitive and specific radioimmunoassays, and RPH activity was measured by a sensitive microassay. The concentrations of each parameter expressed per 10(6) parenchymal or fat-storing cells were, respectively: retinoids, 1.5 and 83.9 micrograms of retinol equivalents; RBP, 138 and 7.4 ng; RPH, 826 and 1152 pmol FFA formed hr-1; CRBP, 470 and 236 ng; and CRABP, 5.6 and 8.7 ng. When these data were expressed on the basis of per unit mass of cellular protein, the concentrations of RPH, CRBP, and CRABP in the fat-storing cells, which contain 10-fold less protein than the large parenchymal cells, were seen to be greatly enriched over parenchymal cells. The parenchymal cells contained approximately 9% of the total retinoids, 98% of the total RBP, 90% of the total RPH activity, 91% of the total CRBP, and 71% of the total CRABP found in the liver. The fat-storing cells accounted for approximately 88% of the total retinoids, 0.7% of the total RBP, 10% of the RPH activity, 8% of the total CRBP, and 21% of the CRABP in the liver. The endothelial and Kupffer cell fractions contained very low levels of all of these parameters. Thus, the large and abundant parenchymal cells account for greater than 70% of the liver's RBP, RPH, CRBP, and CRABP; but the much smaller and less abundant fat-storing cells contain the majority of hepatic retinoids and greatly enriched concentrations of RPH, CRBP, and CRABP.     

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.     

A novel role of fatty acid-binding protein as a vehicle of retinoids

Intracellular transport and storage of retinoids were shown to be conducted by fatty acid-binding protein (FABP). When rat liver cytosol was gel filtrated, retinyl palmitate-binding activity was mainly eluted in the fraction with a Mr. of around 14,000, in which both FABP and cellular retinol-binding protein (CRBP) co-existed. From the binding analysis of purified FABP and CRBP to retinyl palmitate, FABP was found to have a relatively high affinity (Kd = 1.4 X 10(-6) M) to retinyl palmitate, while binding of retinyl palmitate to CRBP was scarcely detectable. By using anti-FABP serum, it was shown that FABP was distributed in organs relating to absorption and storage of retinoids, such as jejunum, ileum, and liver. In liver, the protein was localized in the parenchymal cells and with particularly high concentration in the perisinusoidal cells, probably fat-storing cells.     

Hepatic retinol metabolism. Distribution of retinoids, enzymes, and binding proteins in isolated rat liver cells

The main retinoids and some binding proteins and enzymes involved in retinol metabolism have been quantified in different types of rat liver cells. Hepatic perisinusoidal stellate cells contained 28-34 nmol of retinoids/10(6) cells, and parenchymal liver cells contained 0.5-0.8 nmol of retinoids/10(6) cells, suggesting that as much as 80% of more of total liver retinoids might be stored in stellate cells with the rest stored in parenchymal cells. Isolated endothelial cells and Kupffer cells contained very low levels of retinoids. More than 98% of the retinoids recovered in stellate cells were retinyl esters. Isolated parenchymal and stellate cell preparations both contained considerable retinyl palmitate hydrolase and acyl-CoA:retinol acyltransferase activities. Parenchymal cells accounted for about 75-80% of the total hepatic content of these two enzyme activities, with the rest located in stellate cells. On a cell protein basis, the concentrations of both of these activities were much greater in stellate cells than in parenchymal cells. In contrast, cholesteryl oleate and triolein hydrolase activities were fairly evenly distributed in all types of liver cells. Large amounts of cellular retinol binding proteins were also found in parenchymal and stellate cells. Although parenchymal cells accounted for more than 90% of hepatic cellular retinol binding protein, the concentration of the protein in stellate cells (per unit protein) was 22 X greater than that in parenchymal cells. Stellate cells were also enriched in cellular retinoic acid binding protein. Thus, both parenchymal and stellate cells contain substantial amounts of retinoids and of the enzymes and intracellular binding proteins involved in retinol metabolism. Stellate cells are particularly enriched in these several components.     

Distribution of lecithin-retinol acyltransferase activity in different types of rat liver cells and subcellular fractions

It is now well documented that lecithin-retinol acyltransferase (LRAT) is the physiologically important enzyme activity involved in the esterification of retinol in the liver. However, no information regarding the cellular distribution of this enzyme in the liver is presently available. This study characterizes the distribution of LRAT activity in the different types of rat liver cells. Purified preparations of isolated parenchymal, fat-storing, and Kupffer + endothelial cells were isolated from rat livers and the LRAT activity present in microsomes prepared from each of these cell fractions was determined. The fat-storing cells were found to contain the highest level of LRAT specific activity (383 +/- 54 pmol retinyl ester formed min-1.mg-1 versus 163 +/- 22 pmol retinyl ester formed min-1.mg-1 for whole liver microsomes). The level of LRAT specific activity in parenchymal cell microsomes (158 +/- 53 pmol retinyl ester formed min-1.mg-1) was very similar to LRAT levels in whole liver microsomes. The Kuppfer + endothelial cell microsome fractions were found to contain LRAT, at low levels of activity. These results indicate that the fat-storing cells are very enriched in LRAT but the parenchymal cells also posses significant levels of LRAT activity.     

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.     

Association of cellular retinol-binding protein and several lipid hydrolase activities with a vitamin A-containing high-molecular-weight lipid-protein aggregate from rat liver cytosol

A fluorescent, high-molecular-weight, lipid-protein aggregate was partially isolated from the cytosol fraction of rat liver by gel filtration on columns of Sepharose 4B or 6B. This aggregate was composed of approximately equal parts of protein and of lipid (mainly triglycerides), and was found to contain approximately 19% of the total liver vitamin A (predominantly as retinyl esters). Most of the liver cellular retinol-binding protein (CRBP) was found associated with the fluorescent, lipid-protein aggregate, along with much of the retinyl palmitate hydrolase activity present in the liver cytosol. The lipid-protein aggregate, and its several vitamin A-related components, all displayed an apparent hydrated density between 1.052 and 1.090 in the ultracentrifuge. CRBP in association with the lipid-protein aggregate was not immunoreactive in the CRBP radioimmunoassay. CRBP was, however, released from this aggregate and rendered immunoreactive by addition of detergents (e.g., Triton X-100). Three other lipid hydrolytic activities were also found in association with the lipid-protein aggregate, namely, triolein, cholesteryl oleate, and dipalmitoyl phosphatidylcholine hydrolase activities. These several hydrolytic activities were all found to be stimulated optimally by the addition of either sodium cholate or bovine serum albumin. With the information available, it is not clear whether this lipid-protein aggregate is formed in vitro, during liver homogenization, or whether it represents a specific lipoprotein with a significant functional role that exists in vivo in the liver cell.     

Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate.

Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis-Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 micro m and specific activities between 3 and 8 nmol.min-1.mg-1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.     

Cholate-independent retinyl ester hydrolysis. Stimulation by Apo-cellular retinol-binding protein

Apo-cellular retinol-binding protein (apoCRBP) activated the hydrolysis of endogenous retinyl esters in rat liver microsomes by a cholate independent retinyl ester hydrolase. A Michaelis-Menten relationship was observed between the apoCRBP concentration and the rate of retinol formation, with half-maximum stimulation at 2.6 +/- 0.6 microM (mean +/- S.D., n = 5). Two other retinol-binding proteins, bovine serum albumin and beta-lactoglobulin, acceptors for the rapid and spontaneous hydration of retinol from membranes, had no effect up to 90 microM. These data suggest activation of the hydrolase by apoCRBP directly, rather than by facilitating removal of retinol from membranes. The hydrolase responding was the cholate-independent/cholate-inhibited retinyl ester hydrolase as shown by: 60% inhibition of the apoCRBP effect by 3 mM cholate; apoCRBP enhancement of retinyl ester hydrolysis in liver microsomes that had no detectable cholate-enhanced activity; inhibition of cholate-dependent, but not apoCRBP-stimulated retinyl ester hydrolysis by rabbit anti-rat cholesteryl esterase. Compared to the rate (mean +/- S.D. of [n] different preparations) supported by 5 microM apoCRBP in liver microsomes of 6.7 +/- 3.7 pmol/min/mg protein [10], microsomes from rat lung, kidney, and testes had endogenous retinyl ester hydrolysis rates of 1.8 +/- 0.3 [5], 0.5 +/- 0.2 [3], and 0.3 +/- 0.2 [5] pmol/min/mg protein, respectively. N-Ethylmaleimide and N-tosyl-L-phenylalanine chloromethyl ketone were potent inhibitors of apoCRBP-stimulated hydrolysis with IC50 values of 0.25 and 0.15 mM, respectively, but phenylmethylsulfonyl fluoride and diisopropyl-fluorophosphate were less effective with IC50 values of 1 mM, indicating the importance of imidazole and sulfhydryl groups to the activity. These data provide evidence of a physiological role for the cholate-independent hydrolase in retinoid metabolism and suggest that apoCRBP is a signal for retinyl ester mobilization.     

Retinol esterification in Sertoli cells by lecithin-retinol acyltransferase

Esterification of retinol occurs during the metabolism of vitamin A in the testis. An acyl-CoA:retinol acyltransferase (ARAT) activity has been described for microsomes isolated from testis homogenates. That activity was also observed here in microsomal preparations obtained from cultured Sertoli cells from 20-day-old (midpubertal) rats. ARAT catalyzed the synthesis of retinyl laurate when free retinol and lauroyl-CoA were provided as substrates. However, in the absence of exogenous acyl-CoA, retinol was esterified by a different activity in a manner similar to the lecithin:retinol acyltransferase (LRAT) activity described recently for liver and intestine. Microsomal preparations obtained from enriched Sertoli cell fractions from the adult rat testis had 75-fold higher levels of LRAT than the preparations from midpubertal animals, but ARAT activity was the same in both these preparations. LRAT utilized an endogenous acyl donor and either unbound retinol or retinol complexed with cellular retinol-binding protein (CRBP) to catalyze the synthesis of retinyl linoleate, retinyl oleate, retinyl palmitate, and retinyl stearate. The addition of exogenous dilaurylphosphatidylcholine (DLPC) resulted in the synthesis of retinyl laurate. The esterification from both exogenous DLPC and endogenous acyl donor was inhibited by 2 mM phenylmethanesulfonyl fluoride (PMSF). ARAT activity was not affected by similar concentrations of PMSF. Furthermore, retinol bound to CRBP, a protein known to be present in Sertoli cells, was not an effective substrate for testicular ARAT. When retinol uptake and metabolism were examined in cultured Sertoli cells from 20-day-old rats, the cells synthesized the same retinyl esters that were produced by microsomal LRAT in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical studies on the localization of plasma and of cellular retinol-binding proteins and of transthyretin (prealbumin) in rat liver and kidney

The immunocytochemical localization of cellular retinol-binding protein (CRBP), of plasma retinol-binding protein (RBP), and of plasma transthyretin (TTR) was studied in rat liver and kidney. The studies employed normal rats, retinol-deficient rats, and rats fed excess retinol. Antisera were prepared in rabbits against purified rat CRBP, RBP, and TTR. The primary antibodies and goat anti-rabbit IgG were purified by immunosorbent affinity chromatography, using the respective pure antigen coupled to Sepharose as the immunosorbent. This procedure effectively removed cross-reactive and heterophile antibodies, which permitted the specific staining and localization of each antigen by the unlabeled peroxidase-antiperoxidase method. CRBP was found to be localized in two cell types in the liver, the parenchymal cells and the fat-storing cells. Diffuse cytoplasmic staining for CRBP was seen in all the parenchymal cells. Much more intense staining for CRBP was seen in the fat-storing cells. The prominence of the CRBP-positive fat- storing cells changed markedly with vitamin A status. Thus, these cells were most prominent, and appeared most numerous, in liver from rats fed excess retinol. Both RBP and TTR were localized within liver parenchymal cells. The intensity of RBP staining increased markedly in retinol-deficient rat liver, consistent with previous biochemical observations. With the methods employed, specific staining for RBP or TTR was not seen in cells other than the parenchymal cells. In the kidney, all three proteins (CRBP, RBP, and TTR) were localized in the proximal convoluted tubules of the renal cortex. Staining for RBP was much more intense in normal kidney than in kidney from retinol- deficient rats. These findings reflect the fact that RBP in the tubules represents filtered and reabsorbed RBP. The pattern of specific staining for CRBP among the various tubules was very similar to that seen for RBP on adjacent, serial sections of kidney. The function of CRBP in the kidney is not known.     

Distribution of endogenous retinoids,retinoid binding proteins (RBP,CRABPI) and nuclear retinoid X receptor beta (RXRbeta) in the porcine embryo

Retinoids are important signalling molecules in the development of limbs and in the determination of the anterior-posterior orientation of the embryo. The present study examined the content and distribution of retinoic acid, retinol and retinyl esters in porcine embryos during early gestation (gestation days 22-30) macroscopically and microscopically by its autofluorescence and by HPLC. Macroscopically, the yellowish-greenish autofluorescence characteristic of vitamin A was observed in tissues affected by morphogenesis, such as the limbs, in a spatial and temporal manner. Changes in the intensity of autofluorescence in the limbs paralleled changes in the concentration of retinoids in these structures. In the limbs and the body, retinol, retinyl palmitate, and all-trans-retinoic acid but neither the isomers of all-trans retinoic acid nor other retinoid metabolites were detected. In addition, the distribution of specific retinoid-binding proteins was investigated; these are involved in vitamin A transport, metabolism and signal transduction. Immunoreactive retinol-binding protein as well as cellular retinoic acid binding protein type I were only localised in the mesonephros, while the retinoid X receptor beta was widely distributed in most of the tissues and organs of the embryo throughout the time period investigated. The combination of autofluorescence and HPLC analysis allowed for the first time to attribute the yellowish-greenish autofluorescence in specific regions of the embryo to vitamin A, and offers a method to study the local cellular distribution of retinol and/or retinyl esters as well as their concentrations in embryonic tissues.     

Vitamin A metabolism in the human intestinal Caco-2 cell line

The human intestinal Caco-2 cell line, described as enterocyte-like in a number of studies, was examined for its ability to carry out the metabolism of vitamin A normally required in the absorptive process. Caco-2 cells contained cellular retinol-binding protein II, a protein which is abundant in human villus-associated enterocytes and may play an important role in the absorption of vitamin A. Microsomal preparations from Caco-2 cells contained retinal reductase, acyl-CoA-retinol acyltransferase (ARAT), and lecithin-retinol acyltransferase (LRAT) activities, which have previously been proposed to be involved in the metabolism of dietary vitamin A in the enterocyte. When intact Caco-2 cells were provided with beta-carotene, retinyl acetate, or retinol, synthesis of retinyl palmitoleate, oleate, palmitate, and small amounts of stearate resulted. However, exogenous retinyl palmitate or stearate was not used by Caco-2 cells as a source of retinol for ester synthesis. While there was a disproportionate synthesis of monoenoic fatty acid esters of retinol in Caco-2 cells compared to the retinyl esters typically found in human chylomicrons or the esters normally synthesized in rat intestine, the pattern was consistent with the substantial amount of unsaturated fatty acids, particularly 18:1 and 16:1, found in the sn-1 position of Caco-2 microsomal phosphatidylcholine, the fatty acyl donor for LRAT. Both ARAT and LRAT have been proposed to be responsible for retinyl ester synthesis in the enterocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered retinoid distribution in the repeated epilation (Er) mutant mouse.

The repeated epilation (Er) mutation in mice causes successive loss and regrowth of hair in heterozygotes (Er/+), and blocks orofacial development and epidermal differentiation in lethal homozygotes (Er/Er). Because the mutation affects a systemic factor, because the Er phenotypes resemble exposure to excess retinoids and because retinoids are critical regulators of epithelial differentiation, we examined whether systemic retinoid levels are altered by the Er defect. Liver retinoic acid and retinol were elevated 1.5- and 3.5-fold, respectively, in adult heterozygotes (Er/+) compared to normal (+/+) animals. Retinyl palmitate was increased 2-fold in heterozygous skin and 3-fold in kidney, but the retinol level in plasma was only half that of normal animals. Newborn heterozygous liver also had nearly 2-fold increased retinoids compared to normal. In contrast, Er/Er newborns had reduced retinoid levels in liver, two-thirds the retinol and 15% the retinyl palmitate compared to normal, but greater than 4-fold elevated levels of retinyl palmitate in the extrahepatic body. Tissue contents of retinol binding protein (RBP), which transports vitamin A from the liver to the remainder of the body, were determined by immunoblotting with anti-mouse RBP. Newborn normal and mutant animals had similar liver microsomal RBP contents. RBP contents in plasma and in liver microsomes were also similar in normal and Er/+ adults despite different retinol contents in the Er/+ tissues. Hair follicles of the Er/+, but not the normal adult, were stained with this antiserum to RPB in the outer root sheath layer. These results strongly suggest that altered retinoid distribution is associated with, and may be responsible for, the altered epithelial differentiation in the Er mutant.     

Acyl-CoA-independent esterification of retinol bound to cellular retinol-binding protein (type II) by microsomes from rat small intestine

Cellular retinol-binding protein (type II) (CRBP(II)), a newly described retinol-binding protein, is present in the small intestinal absorptive cell at high levels. Retinol (vitamin A alcohol) presented as a complex with CRBP(II) was found here to be esterified by microsomal preparations from rat small intestinal mucosa. The esterification observed utilized an endogenous acyl donor(s) and produced retinyl esters containing linoleate, oleate, palmitate, and stearate in a proportion quite similar to that previously reported for retinyl esters in lymph and isolated chylomicrons of rat. No dependence on endogenous or exogenous acyl-CoA could be demonstrated. The apparent Km for retinol-CRBP(II) in the reaction with endogenous acyl donor was 2.4 X 10(-7) M. Retinol presented as a complex with CRBP(II) was esterified more than retinol presented as a complex with cellular retinol-binding protein or retinol-binding protein, two other proteins known to bind retinol in vivo, but about the same as retinol presented bound to bovine serum albumin or beta-lactoglobulin. The ability of protein-bound retinol to be esterified was related to accessibility of the hydroxyl group, as judged by the ability of alcohol dehydrogenase to oxidize the bound retinol. However, whereas retinol bound to CRBP(II) was unavailable for esterification in any acyl-CoA-dependent reaction, retinol bound to bovine serum albumin was rapidly esterified in a reaction utilizing exogenous acyl-CoA. The results suggest that one of the functions of CRBP(II) is to accept retinol after it is absorbed or generated from carotenes in the small intestine and present it to the appropriate esterifying enzyme.     

Decreased hepatic retinyl palmitate hydrolase activity in protein-deficient rats

28-day-old weanling rats were fed a diet containing 3% casein as the only source of protein for eight weeks to induce protein deficiency. When compared to control animals (fed a diet containing 25% casein), these rats had significantly lowered body (5.2-fold reduction) and liver (2.5-fold reduction) weights. The circulatory level of retinol (nmol per ml plasma) as well as retinol (nmol per g tissue) in the liver of these protein-deficient animals were also reduced significantly, although their liver concentration of retinyl palmitate (nmol per g tissue) was comparable to that of the control group. Assay of liver tissue for retinyl palmitate hydrolase activity revealed a 4-fold reduction (compared to that of control animals) of specific enzyme activity (nmol retinol formed per g protein per h). These findings suggest that severe protein deficiency results in a decreased hydrolysis of retinyl esters in the liver, which may be in part responsible for the reduced level of metabolically 'active' retinoids available for normal physiological functions.     

Cellular retinol-binding protein type III is needed for retinoid incorporation into milk

The physiologic role(s) of cellular retinol-binding protein (CRBP)-III, an intracellular retinol-binding protein that is expressed solely in heart, muscle, adipose, and mammary tissue, remains to be elucidated. To address this, we have generated and characterized CRBP-III-deficient (CRBP-III(-/-)) mice. Mice that lack CRBP-III were viable and healthy but displayed a marked impairment in retinoid incorporation into milk. Milk obtained from CRBP-III(-/-) dams contains significantly less retinyl ester, especially retinyl palmitate, than milk obtained from wild type dams. We demonstrated that retinol bound to CRBP-III is an excellent substrate for lecithin-retinol acyltransferase, the enzyme responsible for catalyzing retinyl ester formation from retinol. Our data indicated that the diminished milk retinyl ester levels arise from impaired utilization of retinol by lecithin-retinol acyltransferase in CRBP-III(-/-) mice. Interestingly, CRBP-I and CRBP-III each appeared to compensate for the absence of the other, specifically in mammary tissue, adipose tissue, muscle, and heart. For CRBP-III(-/-) mice, CRBP-I protein levels were markedly elevated in adipose tissue and mammary gland. In addition, in CRBP-I(-/-) mice, CRBP-III protein levels were elevated in tissues that normally express CRBP-III but were not elevated in other tissues that do not normally express CRBP-III. Our data suggested that CRBP-I and CRBP-III share some physiologic actions within tissues and that each can compensate for the absence of the other to help maintain normal retinoid homeostasis. However, under conditions of high demand for retinoid, such as those experienced during lactation, this compensation was incomplete.     

Charge effects on phospholipid monolayers in relation to cell motility

A new sensitive method for the assay of retinyl ester hydrolase in vitro was developed and applied to liver homogenates of 18 young pigs with depleted-to-adequate liver vitamin A reserves. Radioactive substrate was not required, because the formation of retinol could be adequately quantitated by reversed-phase high-performance liquid chromatography. Optimal hydrolase activity was observed with 500 microM retinyl palmitate, 100 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 2 mg/ml Triton X-100 at pH 8.0. The relative rates of hydrolysis of six different retinyl esters by liver homogenate were: retinyl linolenate (100%), myristate (99%), palmitate (47%), oleate (38%), linoleate (31%), and stearate (29%). The enzyme was found primarily in the membrane-containing fractions of liver (59 +/- 3%, S.E.) and kidney (76 +/- 3%), with considerably lower overall activity in kidney (57-375 nmol/h per g of tissue) than in liver (394-1040 nmol/h per g). Retinyl ester hydrolase activity in these pigs was independent of serum retinol values, which ranged from 3 to 24 micrograms/dl, and of liver vitamin A concentrations from 0 to 32 micrograms/g. Pig liver retinyl ester hydrolase differs from the rat liver enzyme in its substrate specificity, bile acid stimulation, and interanimal variability.     

An immortalized rat liver stellate cell line (HSC-T6): a new cell model for the study of retinoid metabolism in vitro

Hepatocytes and hepatic stellate cells play important roles in retinoid storage and metabolism. Hepatocytes process postprandial retinyl esters and are responsible for secretion of retinol bound to retinol-binding protein (RBP) to maintain plasma retinol levels. Stellate cells are the body's major cellular storage sites for retinoid. We have characterized and utilized an immortalized rat stellate cell line, HSC-T6 cells, to facilitate study of the cellular aspects of hepatic retinoid processing. For comparison, we also carried out parallel studies in Hepa-1 hepatocytes. Like activated primary stellate cells, HSC-T6 express myogenic and neural crest cytoskeletal filaments. HSC-T6 cells take up and esterify retinol in a time- and concentration-dependent manner. Supplementation of HSC-T6 culture medium with free fatty acids (up to 300 micrometer) does not affect retinol uptake but does enhance retinol esterification up to 10-fold. RT-PCR analysis indicates that HSC-T6 cells express all 6 retinoid nuclear receptors (RARalpha, -beta, -gamma, and RXRalpha, -beta, -gamma) and like primary stellate cells, HSC-T6 stellate cells express cellular retinol-binding protein, type I (CRBP) but fail to express either retinol-binding protein (RBP) or transthyretin (TTR). Addition of retinol (10(-8)-10(-5) m) or all-trans-retinoic acid (10(-10)-10(-6) m) rapidly up-regulates CRBP expression. Using RAR-specific agonists and antagonists and an RXR-specific agonist, we show that members of the RAR-receptor family modulate HSC-T6 CRBP expression.Thus, HSC-T6 cells display the same retinoid-related phenotype as primary stellate cells in culture and will be a useful tool for study of hepatic retinoid storage and metabolism.     

Properties of liver retinyl ester hydrolase in young pigs

A new sensitive method for the assay of retinyl ester hydrolase in vitro was developed and applied to liver homogenates of 18 young pigs with depleted-to-adequate liver vitamin A reserves. Radioactive substrate was not required, because the formation of retinol could be adequately quantitated by reversed-phase high-performance liquid chromatography. Optimal hydrolase activity was observed with 500 μM retinyl palmitate, 100 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 2 mg/ml Triton X-100 at pH 8.0. The relative rates of hydrolysis of six different retinyl esters by liver homogenate were: retinyl linolenate (100%), myristate (99%), palmitate (47%), oleate (38%), linoleate (31%), and streate (29%). The enzyme was found primarily in the membrane-containing fractions of liver (59±3%, S.E.) and kidney (76±3%), with considerably lower overall activity in kidney (57–375 nmol/h per g of tissue) than in liver (394–1040 nmol/h per g). Retinyl ester hydrolase activity in these pigs was independent of serum retinol values, which ranged from 3 to 24 μg/dl, and of liver vitamin A concentrations from 0 to 32 μg/g. Pig liver retinyl ester hydrolase from the rat liver enzyme in its substrate specificity, bile acid stimulation, and interanimal variability.