The biosynthesis of retinoic acid from retinol by rat tissues in vitro

This report shows that a spectrum of vitamin A-dependent tissues can produce retinoic acid by synthesis in situ, indicates that cellular retinol and retinoic acid binding proteins are not obligatory to retinoic acid synthesis, and provides initial characterization of retinoic acid synthesis by rat tissues. Retinoic acid synthesis from retinol was detected in homogenates of rat testes, liver, lung, kidney, and small intestinal mucosa, but not spleen. Zinc did not stimulate the conversion of retinol into retinoic acid by liver homogenates. Retinoic acid synthesis was localized in cytosol of liver and kidney, where its rate of synthesis from retinol was fourfold (liver) and sevenfold (kidney) slower than from retinal. The synthesis of retinoic acid from retinol required NAD and was not supported by NADP. NADH (0.5 mM) reduced retinoic acid synthesis from retinol, supported by NAD (2 mM), by 50-70%, but was fivefold less potent in reducing retinoic acid synthesis from retinal. Dithiothreitol enhanced the conversion of retinol, but not retinal, into retinoic acid. EDTA inhibited the conversion of retinol into retinoic acid slightly (13%, liver; 29%, kidney). A high ethanol concentration (100 mM), relative to retinoid substrate (10 microM), inhibited retinoic acid synthesis from retinol (liver, 54%; kidney, 30%) and from retinal (30%, liver; 9%, kidney). 4'-(9-Acridinylamino)methansulfon-m-anisidine, an inhibitor of aldehyde oxidase, and disulfiram, a sulfhydryl-group crosslinking agent, were potent inhibitors of retinoic acid synthesis at 10 microM or less, and seemed equipotent in liver and kidney. 4-Methylpyrazole, an inhibitor of ethanol metabolism, also inhibited retinoic acid synthesis from retinol, but was less potent than the former two inhibitors, and affected liver to a greater extent than kidney, particularly with retinal as substrate.     

Microsomes convert retinol and retinal into retinoic acid and interfere in the conversions catalyzed by cytosol

Rat liver microsomes converted retinol into retinal and retinoic acid. The production of retinal was observed over a range of substrate concentrations (10-100 microM), but retinoic acid was detected only at retinol concentrations of 50 microM or higher. At 50 microM retinol, the rate of microsomal retinal production was 2-fold greater than that of cytosol, but the rate of retinoic acid synthesis was 4-fold less than that of cytosol. Retinal was also converted into retinoic acid by rat liver microsomes, but at a rate 2-5% of that catalyzed by cytosol. Microsomes also interfered with the conversion of retinol and retinal into retinoic acid by rat liver cytosol. A 50% decrease in the cytosolic rates of retinoic acid production from retinol or retinal was caused by microsomal to cytosolic protein ratios of 0.1 and 0.5, respectively. Under the incubation conditions, which included NAD in the medium, addition of microsomes to cytosol did not affect the elimination half-life of retinol or retinoic acid, but did decrease the elimination half-life of retinal by 2-fold. These data show that retinal synthesis from retinol does not necessarily reflect retinoic acid synthesis and suggest that liver microsomes sequester free retinol and convert it into retinal primarily for elimination, rather than to serve as substrate for cytosolic retinoic acid synthesis.     

Effect of retinoic acid and apo-RBP on serum retinol concentration in acute renal failure

We recently demonstrated a rapid up-regulation of serum retinol-retinol binding protein-transthyretin concentration in rats with short-term acute renal failure. We examine the effect of retinoic acid and apo-retinol binding protein (apo-RBP) on the up-regulation of serum retinol in renal failure. Injection of retinoic acid (10 micrograms) into rats with acute renal failure or sham-operated rats increased circulatory retinoic acid concentration 29-fold within 2 h but did not influence serum retinol concentration in either group. Injection of a large dose of retinoic acid (100 micrograms) decreased serum retinol concentration in rats with acute renal failure (19%) and sham-operated rats (29%). These results suggest that changes in serum retinoic acid concentration within the near-physiological range have no effect on regulation of hepatic retinol release. Injection of a large dose of retinoic acid may depress serum retinol indirectly via a retinol sparing effect in target tissues. In rats with renal failure the serum retinol concentration, elevated 44-52% above that of sham-operated controls, was also increased to 70-164% above controls by the injection of 52-63 micrograms of apo-RBP. This suggests that circulatory apo-RBP can up-regulate serum retinol. Circulatory apo-RBP may be a positive physiological feedback signal from peripheral tissues for hepatic release of retinol.     

Vitamin A and bone formation. Different responses to retinol and retinoic acid of chick bone cells in organ culture

The response of bone cells in organ culture to retinol and retinoic acid was studied. Both stimulated incorporation of [3H]thymidine into DNA by 16-day embryonic chick calvaria, but the time-course of the responses differed; the peak responses to retinol and retinoic acid occurred at about 18 h and 48 h, respectively. Although retinol inhibited chick bone collagen synthesis retinoic acid had no effect, but it did stimulate non-collagenous protein synthesis, whereas the effect on the latter of retinol was, if anything, inhibitory. When present with retinol, retinoic acid was able to attenuate the inhibitory effect of the former on chick bone collagen synthesis, but preincubation with retinoic acid had no such effect. In neonatal murine calvarial cultures, retinoic acid inhibited collagen synthesis selectively in the same manner as did retinol. The ability of chick osteoblasts to respond differently to retinol and retinoic acid suggests that both forms of the vitamin may have a role in bone formation and that their intracellular models of action may differ although the attenuation response indicates there may be some interaction between the two.     

Formation of retinoic acid from retinol in the rat

1. The formation in vivo of retinoic acid from microgram quantities of intrajugularly administered [15-(14)C]retinol was demonstrated in the rat. 2. Endogenously formed retinoic acid (about 0.1mug./rat) was found in liver, and to a much smaller extent in intestine, 12hr. after retinol administration. 3. Excretion of some of the endogenously formed retinoic acid occurred in the bile of bile-duct-cannulated rats. 4. Excretion of unaltered retinoic acid in the urine of intact rats did not occur even after the intrajugular administration of preformed retinoic acid.     

Pathways of retinol and retinoic acid metabolism in the rat

1. The metabolism of retinoic acid and retinyl acetate labelled with (14)C in various positions was studied after intravenous injection of physiological amounts of these compounds into retinol-deficient rats. 2. Analysis of the resultant radio-activity in the urine, carbon dioxide and faeces led to a postulation of the existence of three major pathways for the metabolism of these two compounds. 3. Evidence is presented that retinoic acid and retinol are metabolized by either the same or at least similar pathways and that retinol becomes oxidized to the carboxyl state before any degradation of the isoprenoid side chain occurs. 4. It is not possible to decide from these data whether retinoic acid is an intermediate in the retinol pathway. 5. Possible sites for the regulation of retinol metabolism are discussed.     

Differences in the action and metabolism between retinol and retinoic acid in B lymphocytes

We have previously reported on the dependency of activated B lymphocytes for retinol. Here we confirm and extend these findings that cells deprived of retinol perish in cell culture within days, displaying neither signs of apoptosis nor of cell cycle arrest. Cell death can be prevented by physiological concentrations of retinol and retinal, but not by retinoic acid or three synthetic retinoic acid analogues. To exclude the possibility that retinoic acid is so rapidly degraded as to escape detection, we have tested its stability in intra- and extracellular compartments. Contrary to expectation, we find that retinoic acid persists for longer (t 1/2 3 d) in cultures than retinol (t 1/2 1 d). Furthermore, despite the use of sensitive trace-labeling techniques, we cannot detect retinoic acid or 3,4-didehydroretinoic acid among retinol metabolites. However, retinol is converted into several new retinoids, one of which has the ability to sustain B cell growth in the absence of an external source of retinol, supporting the notion of a second retinol pathway. We have also determined which of the known retinoid-binding proteins are expressed in B lymphoblastoid cells. According to results obtained with polymerase chain reaction-assisted mRNA detection, they transcribe the genes for cellular retinol- and cellular retinoic acid-binding proteins, for the nuclear retinoic acid receptors, RAR-alpha, -gamma, and RXR-alpha, but not RAR-beta. Our findings that B cells do not synthesize retinoic acid or respond to exogenous retinoic acid on the one hand, but on the other hand convert retinol to a novel bioactive form of retinol, suggest the existence of a second retinoid pathway, distinct from that of retinoic acids.     

Biosynthesis of beta-glucuronides of retinol and of retinoic acid in vivo and in vitro

After the intraportal injection of retinol-6,7-(14)C to rats, the O-ether derivative of retinol, retinyl -glucosiduronate, appears in the bile. Both retinoyl -glucuronide and retinyl -glucosiduronate are also synthesized in vitro when washed rat liver microsomes are incubated with uridine diphosphoglucuronic acid (UDPGA) and either retinoic acid or retinol, respectively. The synthesis of retinoyl -glucuronide was also demonstrated in microsomes of the kidney and in particulate fractions of the intestinal mucosa. The glucuronides were characterized by their UV absorption spectra, by their quenching of UV light or fluorescence under it, by their thin-layer chromatographic behavior in two solvent systems, and by the identification of products released during their hydrolysis by -glucuronidase. With retinoic acid as the substrate, the UDP glucuronyl transferase of rat liver microsomes had a pH optimum of 7.0, a temperature optimum of 38 degrees C, and a marked dependence on the concentrations of both retinoic acid and UDPGA, but was unaffected by a number of possible inhibitors, protective agents, and competitive substrates. The conversion of retinal to retinoic acid and the synthesis of retinoyl -glucuronide from retinoic acid could not be detected in whole homogenates, cell fractions, or outer segments of the bovine retina.     

Separable binding proteins for retinoic acid and retinol in bovine retina.

Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [3H] retinoic acid which could not be effectively displaced by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [3H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder.     

Separable binding proteins for retinoic acid and retinol in bovine retina

Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [³H] retinoic acid which could not be effectively displayed by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [³H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder.     

Effect of retinol and retinoic acid on testosterone production by rat Leydig cells in primary culture

Adult rat Leydig cells, purified by Percoll density gradient centrifugation, were used to determine the effect of retinol and retinoic acid on steroidogenesis. It was found that both retinoic acid and retinol stimulated testosterone production. Although retinol was less potent than retinoic acid, retinol had the greater efficacy. When these retinoids were tested in the presence of a maximal dose of LH, it was found that retinol inhibited LH-stimulated testosterone synthesis whereas retinoic acid had no similar effect. These results demonstrate for the first time that retinol and retinoic acid have a direct effect on Leydig cell steroidogenesis in culture suggesting that retinoids play a role in the maintenance and regulation of Leydig cell function.     

Effects of retinoic acid on the concentrations of radioactive metabolites of retinol in tissues of rats maintained on a retinol-deficient diet

The effects of feeding retinoic acid for 2 and 6 days on the metabolism of labeled retinol in tissues of rats maintained on a vitamin A deficient diet was studied. The metabolites of retinol were analyzed by high performance liquid chromatography. Feeding retinoic acid for 2 days significantly reduced the blood retinol and retinyl ester levels without affecting the vitamin A content of the liver. In intestine and testis the content of labeled retinoic acid was decreased significantly by dietary retinoic acid. Addition of retinoic acid to the diet for 6 days resulted, in addition to decreased blood retinol and retinyl ester values, in an increase in the retinyl ester values in the liver. The accumulation of retinyl ester in the retinoic acid fed rat liver was accompanied by an absence of labeled retinoic acid. Kidney tissue was found to contain the highest levels of labeled retinoic acid, retinol, and retinyl esters; dietary retinoic acid did not alter the concentrations of these retinoids in the kidney during the experimental period. Since kidney retained more vitamin A when the liver vitamin A was low and also dietary retinoic acid did not affect the concentrations of radioactive retinoic acid in the kidney, it is suggested that the kidney may play a major role in the production of retinoic acid from retinol in the body.     

Comparison of the effects of retinol and retinoic acid on postimplantation rat embryos in vitro

Rat embryos were explanted on day 8 or 9 of pregnancy and cultured for up to 48 hours in serum containing added retinol (vitamin A), retinoic acid (vitamin A acid), or absolute ethanol. They were examined morphologically and their protein content determined. Retinoic acid was more teratogenic and growth-retarding than retinol. Electron microscopy of embryos cultured for 30 minutes or one hour revealed that both forms of vitamin A brought about similar ultrastructural effects on the embryonic cells; however, the abnormally large intracellular lipid droplets observed in a previous study following exposure to retinol in vitro and retinyl palmitate in vivo were not observed in embryos exposed to retinoic acid. It is possible that the differential teratogenicity may be due to the inability of the embryonic cells to convert and store retinoic acid in a less teratogenic form.     

A novel short-chain alcohol dehydrogenase from rats with retinol dehydrogenase activity,cyclically expressed in uterine epithelium

Retinoic acid is necessary for the maintenance of many lining epithelia of the body, such as the epithelium of the luminal surface of the uterus. Administration of estrogen to prepubertal rats induces in these epithelial cells the ability to synthesize retinoic acid from retinol, coincident with the appearance of cellular retinoic acid-binding protein, type two, which is normally present in these cells only at estrus in the mature, cycling animal. Here, we report the isolation, from a cDNA library prepared from uterine mRNA collected at the estrous stage and from a rat mammary adenocarcinoma cell line, of a cDNA that encodes a novel retinol dehydrogenase. A member of the short-chain alcohol dehydrogenase family, the encoded enzyme was capable of metabolizing retinol to retinal when expressed in cells after transfection of its cDNA. When cotransfected with the cDNA of human aldehyde 6, a known retinaldehyde dehydrogenase, the transfected cells synthesized retinoic acid from retinol. Immunohistochemical analysis revealed that the protein was present in the uterine lining epithelium of the mature animal only at estrus, coincident with the presence of cellular retinol-binding protein and cellular retinoic acid-binding protein, type two. Consequently, this novel short-chain alcohol dehydrogenase is an excellent candidate for the retinol dehydrogenase that catalyzes the first step in retinoic acid biosynthesis that occurs in uterine epithelial cells.     

Holocellular retinol binding protein as a substrate for microsomal retinal synthesis

Holocellular retinol binding protein (holo-CRBP) was substrate for retinal synthesis at physiological pH with microsomes prepared from rat liver, kidney, lung, and testes. Four observations indicated that retinal synthesis was supported by holo-CRBP directly, rather than by the unbound retinol in equilibrium with CRBP. First, the rate of retinal synthesis with holo-CRBP exceeded the rate that was observed from the concentration of unbound retinol in equilibrium with CRBP. Second, NADP was the preferred cofactor only with holo-CRBP, supporting a rate about 3-fold greater than that of NAD. In contrast, with unbound retinol as substrate, similar rates of retinal formation were supported by either NAD or NADP. Third, the rate of retinal synthesis was not related to the decrease in the concentration of unbound retinol in equilibrium with holo-CRBP caused by increasing the concentration of apo-CRBP. Fourth, the rate of retinal synthesis increased with increases in the concentration of holo-CRBP as a fixed concentration of unbound retinol was maintained. This was achieved by increasing both apo-CRBP and holo-CRBP, but keeping constant the ratio apo-CRBP/holo-CRBP. Retinal formation from holo-CRBP displayed typical Michaelis-Menten kinetics with a Km about 1.6 microM, less than the physiological retinal concentration of 4-10 microM in the livers of rats fed diets with recommended vitamin A levels. The Vmax for retinal formation from holo-CRBP was 14-17 pmol min-1 (mg of protein)-1, a rate sufficiently high to generate adequate retinal to contribute significantly to retinoic acid synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation and enterohepatic circulation of metabolites of retinol and retinoic acid in bile duct-cannulated rats

Four hours after intraportal injection of retinoic acid-(14)C into bile duct-cannulated rats, less than 10% of the radioactivity was recovered in the liver, intestine, and kidneys. Within 6 hr, 40% of the radioactivity had appeared in bile. When suspensions of retinol-(14)C or retinal were similarly injected, 25-35% of the dose was excreted in bile within 24 hr and equivalent amounts were deposited in the liver as retinol ester. The isolated perfused liver also produced these bile metabolites and is probably the major site of their formation in vivo. The intestine may metabolize retinoic acid, however, since some metabolites were found in the intestinal wall and lumen, even in bile duct-cannulated rats. The bile metabolites of retinol-(14)C and retinoic acid-(14)C undergo extensive enterohepatic circulation. The bile radioactivity was not volatilized on boiling at acid pH, was not present in digitonin-precipitated sterols, and did not migrate with bile salts on reversed-phase paper chromatography. Anion-exchange chromatography resolved the metabolites of bile into three fractions containing nonionic compounds, acidic substances like retinoic acid, and more polar acidic derivatives.     

Biosynthesis of all-trans-retinoic acid from retinal. Recognition of retinal bound to cellular retinol binding protein (type I) as substrate by a purified cytosolic dehydrogenase.

An NAD-dependent rat liver cytosolic dehydrogenase accepted as substrate retinal generated in situ by microsomes from retinol bound to excess CRBP (cellular retinol binding protein, type I). This activity, which was not retained by anion-exchange chromatography at pH 9.15, was designated P1. P1 activity increased 2.5-fold, with no statistically significant change in its K or Hill coefficient, in liver cytosol from rats fed a retinoid-deficient diet. Orally dosed retinoic acid partially suppressed the increase. Activities chromatographically similar to hepatic P1 were observed in cytosols from rat kidney and testes. P1, purified from rat liver cytosol, had a pI of approximately 8.3, migrated as a tetramer (214 kDa) on a Sephadex G-200 column, and had a subunit molecular mass of 55 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With free retinal it catalyzed a maximum rate of retinoic acid synthesis of 265 nmol/min/mg of protein and exhibited allosteric kinetics with a K of 0.76 +/- 0.35 microM and a Hill coefficient of 1.5 +/- 0.13 (mean +/- S.D., n = 4). Substrate inhibition was noted with retinal concentrations greater than 6 microM. The purified enzyme not only recognized retinal generated by microsomes as substrate, but also recognized retinal bound to CRBP. The rates of retinoic acid synthesis from CRBP-retinal, with a series of increasing apoCRBP concentrations, exceeded the rates that would be supported by the free retinal present. The CRBP-retinal complex exhibited allosteric kinetics (K, 0.13 microM; Hill coefficient, 1.75; averages of duplicates) in the presence of excess apoCRBP (the ratio total CRBP/total retinal at each concentration of retinal was 2). This enzyme is likely to play a significant role in retinoic acid synthesis in vivo, because it participates in the synthesis of retinoic acid from a physiologically occurring form of retinol (holoCRBP), reflects retinoid status, and is distributed in extrahepatic tissues in addition to liver. These results also suggest a novel role for CRBP in retinoid metabolism, facilitating the conversion of retinal into retinoic acid.     

Biogenesis of retinoic acid from beta-carotene. Differences between the metabolism of beta-carotene and retinal

The ability of beta-carotene to serve as precursor to retinoic acid was examined in vitro with cytosol prepared from rat tissues. The rate of retinoic acid synthesis from 10 microM beta-carotene ranged from 120 to 224 pmol/h/mg of protein with intestinal cytosol, and from 344 to 488 pmol/h/mg of protein with cytosols prepared from kidney, lung, testes, and liver. Retinol generated during beta-carotene metabolism was not the major substrate for retinoic acid synthesis. At low substrate concentrations (2.5 microM), the rates of retinoic acid synthesis in intestinal cytosol from beta-carotene or retinol were equivalent, and at higher concentrations (10 microM) the rates of retinoic acid synthesis from beta-carotene or retinol in intestine, testes, lung, and kidney were comparable. Thus, beta-carotene metabolism may be an important source of retinoic acid in retinoid target tissues, particularly in species such as humans that are capable of accumulating high concentrations of tissue carotenoids. Retinal, considered an initial retinoid product of beta-carotene metabolism, was not detected as a product of beta-carotene metabolism in vitro. A ratio of retinol and retinoic acid different from that observed during beta-carotene metabolism in vitro was observed with incubations of retinal under identical conditions. These data indicated that beta-carotene metabolism is not merely a simple process of producing retinal and releasing it into solution to be metabolized independently.     

Retinoic acid rapidly induces lung cellular retinol-binding protein mRNA levels in retinol deficient rats

Retinol deficiency resulted in decreased mRNA levels for cellular retinol-binding protein (CRBP) in the lungs and the testes. The level of lung CRBP mRNA increased 2.3-fold one hour after oral administration of retinoic acid to retinol deficient rats. In contrast, testicular CRBP mRNA level was not influenced. Our data indicate that retinoic acid regulates CRBP mRNA level in the whole animal and this rapid effect suggests a role for CRBP in the mechanism of vitamin A action at genomic level.     

Retinol dehydrogenase 10 but not retinol/sterol dehydrogenase(s) regulates the expression of retinoic acid-responsive genes in human transgenic skin raft culture

Retinoic acid is essential for skin growth and differentiation, and its concentration in skin is controlled tightly. In humans, four different members of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins were proposed to catalyze the rate-limiting step in the biosynthesis of retinoic acid (the oxidation of retinol to retinaldehyde). Epidermis contains at least three of these enzymes, but their relative importance for retinoic acid biosynthesis and regulation of gene expression during growth and differentiation of epidermis is not known. Here, we investigated the effect of the four human SDRs on retinoic acid biosynthesis, and their impact on growth and differentiation of keratinocytes using organotypic skin raft culture model of human epidermis. The results of this study demonstrate that ectopic expression of retinol dehydrogenase 10 (RDH10, SDR16C4) in skin rafts dramatically increases proliferation and inhibits differentiation of keratinocytes, consistent with the increased steady-state levels of retinoic acid and activation of retinoic acid-inducible genes in RDH10 rafts. In contrast, SDRs with dual retinol/sterol substrate specificity, namely retinol dehydrogenase 4 (RoDH4, SDR9C8), RoDH-like 3α-hydroxysteroid dehydrogenase (RL-HSD, SDR9C6), and RDH-like SDR (RDHL, SDR9C4) do not affect the expression of retinoic acid-inducible genes but alter the expression levels of several components of extracellular matrix. These results reveal essential differences in the metabolic contribution of RDH10 versus retinol/sterol dehydrogenases to retinoic acid biosynthesis and provide the first evidence that non-retinoid metabolic products of retinol/sterol dehydrogenases affect gene expression in human epidermis.