Acidic retinoids synergize with vitamin A to enhance retinol uptake and STRA6, LRAT, and CYP26B1 expression in neonatal lung
2009; Elsevier BV; Volume: 51; Issue: 2 Linguagem: Inglês
10.1194/jlr.m001222
ISSN1539-7262
Autores Tópico(s)Retinopathy of Prematurity Studies
ResumoVitamin A (VA) is essential for fetal lung development and postnatal lung maturation. VA is stored mainly as retinyl esters (REs), which may be mobilized for production of retinoic acid (RA). This study was designed 1) to evaluate several acidic retinoids for their potential to increase RE in the lungs of VA-supplemented neonatal rats, and 2) to determine the expression of retinoid homeostatic genes related to retinol uptake, esterification, and catabolism as possible mechanisms. When neonatal rats were treated with VA combined with any one of several acidic retinoids (RA, 9-cis-RA, or Am580, a stable analog of RA), lung RE increased ∼5–7 times more than after an equal amount of VA alone. Retinol uptake and esterification during the period of absorption correlated with increased expression of both STRA6 (retinol-binding protein receptor) and LRAT (retinol esterification), while a reduction in RE after 12 h in Am580-treated, VA-supplemented rats correlated with a strong and persistent increase in CYP26B1 (RA hydroxylase). We conclude that neonatal lung RE can be increased synergistically by VA combined with both natural and synthetic acidic retinoids, concomitant with induction of the dyad of STRA6 and LRAT. However, the pronounced and prolonged induction of CYP26B1 by Am580 may counteract lung RE accumulation after the absorption process is completed. Vitamin A (VA) is essential for fetal lung development and postnatal lung maturation. VA is stored mainly as retinyl esters (REs), which may be mobilized for production of retinoic acid (RA). This study was designed 1) to evaluate several acidic retinoids for their potential to increase RE in the lungs of VA-supplemented neonatal rats, and 2) to determine the expression of retinoid homeostatic genes related to retinol uptake, esterification, and catabolism as possible mechanisms. When neonatal rats were treated with VA combined with any one of several acidic retinoids (RA, 9-cis-RA, or Am580, a stable analog of RA), lung RE increased ∼5–7 times more than after an equal amount of VA alone. Retinol uptake and esterification during the period of absorption correlated with increased expression of both STRA6 (retinol-binding protein receptor) and LRAT (retinol esterification), while a reduction in RE after 12 h in Am580-treated, VA-supplemented rats correlated with a strong and persistent increase in CYP26B1 (RA hydroxylase). We conclude that neonatal lung RE can be increased synergistically by VA combined with both natural and synthetic acidic retinoids, concomitant with induction of the dyad of STRA6 and LRAT. However, the pronounced and prolonged induction of CYP26B1 by Am580 may counteract lung RE accumulation after the absorption process is completed. Vitamin A (VA) is an essential micronutrient that is required for fetal lung development and alveolar septation in the postnatal period (1Roth-Kleiner M. Post M. Similarities and dissimilarities of branching and septation during lung development.Pediatr. Pulmonol. 2005; 40: 113-134Google Scholar, 2Massaro D. Massaro G.D. Pre- and postnatal lung development, maturation, and plasticity - Invited review: Pulmonary alveoli: formation, the "call for oxygen," and other regulators.Am. J. Physiol. Lung Cell. Mol. Physiol. 2002; 282: L345-L358Google Scholar, 3Maden M. Hind M. Retinoic acid in alveolar development, maintenance and regeneration.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2004; 359: 799-808Google Scholar). The active metabolite of VA, retinoic acid (RA), fulfills nearly all of the functions of VA, but unlike VA, which is stored in tissues as retinyl ester (RE) and mobilized to produce RA (4Ross A.C. Harrison E.H. Vitamin A: nutritional aspects of retinoids and carotenoids.in: Zempleni J. Rucker R.B McCormick D.B. Suttie J.W. Handbook of Vitamins. Taylor & Francis Group, Boca Raton, FL2007: 1-40Google Scholar), no significant amount of RA is stored and the pool of RA turns over rapidly (5Rigas J.R. Francis P.A. Muindi J.R.F. Kris M.G. Huselton C. DeGrazia F. Orazem J.P. Young C.W. Warrell Jr, R.P. Constitutive variability in the pharmacokinetics of the natural retinoid, all-trans-retinoic acid, and its modulation by ketoconazole.J. Natl. Cancer Inst. 1993; 85: 1921-1926Google Scholar). RA and other retinoids have been shown to enhance alveolar septation in neonatal rats and mice (as reviewed in Refs. 2Massaro D. Massaro G.D. Pre- and postnatal lung development, maturation, and plasticity - Invited review: Pulmonary alveoli: formation, the "call for oxygen," and other regulators.Am. J. Physiol. Lung Cell. Mol. Physiol. 2002; 282: L345-L358Google Scholar, 3Maden M. Hind M. Retinoic acid in alveolar development, maintenance and regeneration.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2004; 359: 799-808Google Scholar, 6Hind M. Corcoran J. Maden M. Pre- and postnatal lung development, maturation, and plasticity - temporal/spatial expression of retinoid binding proteins and RAR isoforms in the postnatal lung.Am. J. Physiol. Lung Cell. Mol. Physiol. 2002; 282: L468-L476Google Scholar, 7Hind M. Maden M. Retinoic acid induces alveolar regeneration in the adult mouse lung.Eur. Respir. J. 2004; 23: 20-27Google Scholar) and in some cases to improve lung repair after injury in adults (3Maden M. Hind M. Retinoic acid in alveolar development, maintenance and regeneration.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2004; 359: 799-808Google Scholar, 8Belloni P.N. Garvin L. Mao C.P. Bailey-Healy I. Leaffer D. Effects of all-trans-retinoic acid in promoting alveolar repair.Chest. 2000; 117: 235S-241SGoogle Scholar, 9Massaro D. Massaro G.D. Toward therapeutic pulmonary alveolar regeneration in humans.Proc. Am. Thorac. Soc. 2006; 3: 709-712Google Scholar). Most of the VA present in the lungs is in the form of RE (10Shenai J.P. Chytil F. Vitamin A storage in lungs during perinatal development in the rat.Biol. Neonate. 1990; 57: 126-132Google Scholar, 11Shenai J.P. Chytil F. Effect of maternal vitamin-A administration on fetal lung vitamin-A stores in the perinatal rat.Biol. Neonate. 1990; 58: 318-325Google Scholar, 12Ross A.C. Ambalavanan N. Zolfaghari R. Li N-q. Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in neonatal rats.J. Lipid Res. 2006; 47: 1844-1851Google Scholar). In full-term infants, a process of significant RE accumulation in the lungs has begun from the third trimester of fetal life, after which the stored RE becomes quickly depleted during late gestation and early postnatal life (10Shenai J.P. Chytil F. Vitamin A storage in lungs during perinatal development in the rat.Biol. Neonate. 1990; 57: 126-132Google Scholar). By contrast, preterm infants often have low VA status at birth (13Shenai J.P. Chytil F. Jhaveri A. Stahlman M.T. Plasma vitamin A and retinol-binding protein in premature and term neonates.J. Pediatr. 1981; 99: 302-305Google Scholar, 14Shenai J.P. Rush M.G. Stahlman M.T. Chytil F. Vitamin A supplementation and bronchopulmonary dysplasia–revisited.J. Pediatr. 1992; 121: 399-401Google Scholar, 15Mactier H. Weaver L.T. Vitamin A and preterm infants: what we know, what we don't know, and what we need to know.Arch. Dis. Child. Fetal Neonatal Ed. 2005; 90: F103-F108Google Scholar), which may contribute to poor lung maturation and increased susceptibility to respiratory diseases (16Chan V. Cheeseman P. Gamsu H.R. Vitamin A status in preterm and term infants at birth.J. Perinat. Med. 1993; 21: 59-62Google Scholar). Therefore, ways to improve RE storage in the lungs in the postnatal period could be useful clinically for supporting retinoid-requiring metabolic functions and aiding postnatal lung development. Previously, we tested a combination of VA (retinol) and RA, referred to as VARA (10:1 molar mixture of VA and RA) as an oral supplement for promoting lung RE formation (12Ross A.C. Ambalavanan N. Zolfaghari R. Li N-q. Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in neonatal rats.J. Lipid Res. 2006; 47: 1844-1851Google Scholar). Lung RE increased synergistically, at least 4-fold more than for an equal amount of VA alone (17Ross A.C. Li N. Wu L. The components of VARA, a nutrient-metabolite combination of vitamin A and retinoic acid, act efficiently together and separately to increase retinyl esters in the lungs of neonatal rats.J. Nutr. 2006; 136: 2803-2807Google Scholar). As RA is not reduced to retinol in vivo and thus is not a substrate for tissue RE synthesis, the increase in RE in the lungs implies that RA plays a regulatory role in this organ, in some manner facilitating RE formation. The synergistic effect of VARA was selective for the lungs, as RE formation in the liver did not differ between VARA and an equal dose of VA only (12Ross A.C. Ambalavanan N. Zolfaghari R. Li N-q. Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in neonatal rats.J. Lipid Res. 2006; 47: 1844-1851Google Scholar, 17Ross A.C. Li N. Wu L. The components of VARA, a nutrient-metabolite combination of vitamin A and retinoic acid, act efficiently together and separately to increase retinyl esters in the lungs of neonatal rats.J. Nutr. 2006; 136: 2803-2807Google Scholar). In a metabolic study using [3H]retinol to trace the uptake of newly absorbed retinol, we found that RA served to direct more of the [3H]retinol tracer and, thus, the oral VA supplement, into the neonatal lung (12Ross A.C. Ambalavanan N. Zolfaghari R. Li N-q. Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in neonatal rats.J. Lipid Res. 2006; 47: 1844-1851Google Scholar). The inclusion of RA in the diet of adult rats also increased retinol uptake kinetics into the lung (18Cifelli C.J. Green J.B. Green M.H. Dietary retinoic acid alters vitamin A kinetics in both the whole body and in specific organs of rats with low vitamin A status.J. Nutr. 2005; 135: 746-752Google Scholar). However, the mechanisms involved in the VARA synergy are still unknown. Of the many genes that are regulated in vivo by RA, several play prominent roles in retinoid homeostasis (19Ross A.C. Zolfaghari R. Weisz J. Vitamin A: recent advances in the biotransformation, transport, and metabolism of retinoids.Curr. Opin. Gastroenterol. 2001; 17: 184-192Google Scholar). Lecithin:retinol acyltransferase (LRAT) and RA hydroxylases of the CYP26 family of cytochrome P450 genes, which catalyze the esterification of retinol and the oxidation of RA, respectively, are known to be regulated by RA in certain tissues (20Ross A.C. Retinoid production and catabolism: role of diet in regulating retinol esterification and retinoic acid oxidation.J. Nutr. 2003; 133: 291S-296SGoogle Scholar). The expression of these enzymes varies with VA status and appears to provide a level of control over VA metabolism (20Ross A.C. Retinoid production and catabolism: role of diet in regulating retinol esterification and retinoic acid oxidation.J. Nutr. 2003; 133: 291S-296SGoogle Scholar). During VA sufficiency, the continued presence of RA, a signal of high VA status, maintains the expression of LRAT (20Ross A.C. Retinoid production and catabolism: role of diet in regulating retinol esterification and retinoic acid oxidation.J. Nutr. 2003; 133: 291S-296SGoogle Scholar). CYP26A1 and CYP26B1 also increase in some tissues when the concentration of RA rises (21Petkovich P.M. Retinoic acid metabolism.J. Am. Acad. Dermatol. 2001; 45: S136-S142Google Scholar). Conversely, in the situation of VA deficiency, when the concentration of RA is very low, LRAT is downregulated (22Randolph R.K. Ross A.C. Vitamin A status regulates hepatic lecithin:retinol acyltransferase activity in rats.J. Biol. Chem. 1991; 266: 16453-16457Google Scholar), and CYP26 is maintained at a very low level (23Yamamoto Y. Zolfaghari R. Ross A.C. Regulation of CYP26 (Cytochrome P450RAI) mRNA expression and retinoic acid metabolism by retinoids and dietary vitamin A in liver of mice and rats.FASEB J. 2000; 14: 2119-2127Google Scholar, 24Wang Y. Zolfaghari R. Ross A.C. Cloning of rat cytochrome P450RAI (CYP26) cDNA and regulation of its gene expression by all-trans-retinoic acid in vivo.Arch. Biochem. Biophys. 2002; 401: 235-243Google Scholar). Therefore, to a certain extent, the self-regulatory mechanisms involving RA render the body able to modulate its retinol metabolism and thus to avoid both VA deficiency and toxicity. Another protein that may contribute to retinoid homeostasis is STRA6 (stimulated by retinoic acid gene 6), a transmembrane receptor for retinol-binding protein (RBP), and mediator of retinol uptake from plasma and extracellular fluid into cells (25Kawaguchi R. Yu J. Honda J. Hu J. Whitelegge J. Ping P. Wiita P. Bok D. Sun H. A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A.Science. 2007; 315: 820-825Google Scholar). STRA6 is expressed at high levels in the retina and at lower levels in a variety of embryonic and adult cells or organs (26Bouillet P. Sapin V. Chazaud C. Messaddeq N. Décimo D. Dollé P. Chambon P. Developmental expression pattern of Stra6, a retinoic acid-responsive gene encoding a new type of membrane protein.Mech. Dev. 1997; 63: 173-186Google Scholar), including adult lung tissue (27Pasutto F. Sticht H. Hammersen G. Gillessen-Kaesbach G. Fitzpatrick D.R. Nürnberg G. Brasch F. Schirmer-Zimmermann H. Tolmie J. Chitayat D. et al.Mutations in STRA6 cause a broad spectrum of malformations including anophthalmia, congenital heart defects, diaphragmatic hernia, alveolar capillary dysplasia, lung hypoplasia, and mental retardation.Am. J. Hum. Genet. 2007; 80: 550-560Google Scholar). But whether the STRA6 gene is expressed in the neonatal lung and involved in VA uptake in the postnatal period is still unclear. In this study, we first determined whether acidic retinoids besides all-trans-RA can produce the VARA synergy. We then tested the hypothesis that RA and Am580, a retinobenzoic acid analog of RA, which is known both for its resistance to metabolism and its selective activation of the nuclear receptor RAR-α (28Hashimoto Y. Kagechika H. Shudo K. Expression of retinoic acid receptor genes and the ligand-binding selectivity of retinoic acid receptors (RAR'S).Biochem. Biophys. Res. Commun. 1990; 166: 1300-1307Google Scholar, 29Kagechika H. Novel synthetic retinoids and separation of the pleiotropic retinoidal activities.Curr. Med. Chem. 2002; 9: 591-608Google Scholar), can acutely alter neonatal lung retinoid metabolism. We thus determined changes in lung RE formation, [3H]retinol uptake, and the expression of some important retinoid homeostatic genes, including LRAT, CYP26A1, CYP26B1, and STRA6, in neonatal rats treated with VA, RA, VARA, Am580, or VA+Am580 (VAAm). VA, in the form of all-trans-retinyl palmitate, all-trans-RA (at-RA), and 9-cis-RA, was purchased from Sigma-Aldrich (St. Louis, MO). Am580 was a gift of H. Kagechika, University of Tokyo. VARA and related treatments were prepared as described previously (12Ross A.C. Ambalavanan N. Zolfaghari R. Li N-q. Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in neonatal rats.J. Lipid Res. 2006; 47: 1844-1851Google Scholar). The dose concentration of VA alone and after combination with an acidic retinoid was 0.05 M, and the dose concentration of the acidic retinoids (all-trans-RA, 9-cis-RA, or Am580) was 0.005 M. In practice, solutions were prepared by weight rather than volume for accuracy and were confirmed by UV spectrophotometry. Canola oil only was used as placebo (control). Animal procedures were approved by the Institutional Animal Use and Care Committee, Pennsylvania State University. In each of three experiments, Sprague-Dawley rat pups with their dams were assigned to treatments across litters, with pups of both sexes included in each treatment group. The average body weight of each group was similar (data not shown). Before each treatment, the pups were weighed and the dose adjusted to 0.4 µl/g body weight (20 nmol retinol and 2 nmol of acidic retinoid, depending on treatment group, per gram of body weight). The dose was delivered directly into the pup's mouth by a micropipette. In experiment 1 (3 day study, n = 4/group) 5 day old neonates were treated once a day for 3 days with oil (control), VA alone, RA, 9-cis-RA, both isomers mixed in a 1:1 ratio, or VA combined with each the same acidic retinoids. Twenty-four hours after the last treatment, neonates were euthanized and tissues collected (12Ross A.C. Ambalavanan N. Zolfaghari R. Li N-q. Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in neonatal rats.J. Lipid Res. 2006; 47: 1844-1851Google Scholar). In experiments 2 and 3 (acute studies lasting 6 and 12 h), 6–7 day old pups were treated with oil (control), VA alone, RA alone, Am580 alone, VA combined with RA (VARA), or with Am580 (VAAm). In the 12 h study, 2 µCi of [3H]retinol, prepared as previously described (12Ross A.C. Ambalavanan N. Zolfaghari R. Li N-q. Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in neonatal rats.J. Lipid Res. 2006; 47: 1844-1851Google Scholar), was administered orally just after each treatment dose to provide a tracer for the tissue uptake and metabolism of newly absorbed retinol. At designated times, pups were killed with carbon dioxide. Heparinized blood was collected from the vena cava or heart for preparation of plasma, and the lungs and liver were removed, trimmed, weighed, and frozen immediately in liquid nitrogen. Tissue lipids were extracted by the procedure of Folch, Lees, and Sloane Stanley (30Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Google Scholar). A portion of the extracts underwent an alkaline hydrolysis to convert RE to retinol and, after addition of a known amount of an internal standard, trimethylmethoxyphenyl-retinol, samples in 100 µl of methanol were subjected to analysis by reverse-phase HPLC (31Ross A.C. Separation and quantitation of retinyl esters and retinol by high-performance liquid chromatography.Methods Enzymol. 1986; 123: 68-74Google Scholar), monitored by a Waters 960 photodiode array detector (Milford, MA). The areas of the peaks for trimethylmethoxyphenyl-retinol and retinol were analyzed by Millenium-32 (Waters) software, and tissue total retinol concentrations were calculated. For some samples, the concentration of tissue RE was determined without saponification (31Ross A.C. Separation and quantitation of retinyl esters and retinol by high-performance liquid chromatography.Methods Enzymol. 1986; 123: 68-74Google Scholar). Because RE constituted >90% of total retinol for each treatment group, we have referred to total retinol as RE in the figures and text, unless otherwise indicated. Extracts of lung and liver total lipids, prepared as described above, were evaporated and subjected to liquid scintillation spectrometry to determine the total 3H (percent of dose) in the lungs and liver 12 h after dosing. Another portion of the organic solvent extract was dried, redissolved in hexanes, subjected to alumina column chromatography, and counted for [3H]RE to determine uptake as the percentage of the oral dose (12Ross A.C. Ambalavanan N. Zolfaghari R. Li N-q. Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in neonatal rats.J. Lipid Res. 2006; 47: 1844-1851Google Scholar). The upper aqueous phases from the Folch wash were pooled, and a small portion was counted to assess the formation of aqueous [3H]labeled polar metabolites of retinol (32Ross A.C. Retinol esterification by mammary gland microsomes from the lactating rat.J. Lipid Res. 1982; 23: 133-144Google Scholar). The percentage of the oral dose in each tissue fraction was then calculated. The aqueous phase contributed <0.05 and 0.15% of the total 3H in lung and liver, respectively, and thus only total 3H and the percentage of 3H as [3H]RE are reported. Total RNA from the lungs of individual pups was extracted using a guanidine extraction method, and cDNA was prepared using reverse transcriptase (33Zolfaghari R. Ross A.C. Lecithin:retinol acyltransferase from mouse and rat liver: cDNA cloning and liver-specific regulation by dietary vitamin A and retinoic acid.J. Lipid Res. 2000; 41: 2024-2034Google Scholar). The equivalent of 0.05 µg RNA, as cDNA, was used for real-time PCR analysis. Primers designed to detect mRNA expression were as follows: rat LRAT (GenBank accession number AF255060), 5′-ATAGGATCCTGACCAACACTACATCCTCTC-3′ (forward) and 5′-ATTCTCGAGTCTAAGTTTATTGAAACCCCAGA-3′ (reverse); rat CYP26B1 (NM_181087), 5′-TTGAGGGCTTGGAGTTGGT-3′ (forward) and 5′-AACGTTGCCATACTTCTCGC-3′ (reverse); rat CYP26A1 (DQ266888), 5′-GTGCCAGTGATTGCTGAAGA-3′ (forward) and 5′-GGAGGTGTCCTCTGGATGAA-3′ (reverse); rat STRA6 (NM_0010029924.1), 5′-CCGATCCTGGACAGTTCCTA-3′ (forward) and 5′-CCACCTGGTAAGTGGCTGTT -3′ (reverse). The mRNA expression level of each sample was corrected by calculating mRNA-to-ribosomal 18S RNA ratio. Data were normalized to the average value for the control group, set at 1.00, prior to statistical analysis. Data are presented as the group mean ± SE. Depending on the experiment, differences were tested by one-factor ANOVA or two-factor ANOVA followed by Fisher's protected least significant difference test and least squares means test. When variances were unequal, data were transformed to log10 values before statistic analysis. Differences with P < 0.05 were considered significant. All-trans-RA is considered the major form of RA with endogenous activity as a ligand for nuclear receptors of the RAR family (RAR-α, RAR-β, and RAR-γ) (34Bastien J. Rochette-Egly C. Nuclear retinoid receptors and the transcription of retinoid-target genes.Gene. 2004; 328: 1-16Google Scholar). Although the status of 9-cis-RA as an endogenously produced retinoid is uncertain, it is known as an effective activator for receptors of the RXR family, when added exogenously. In some situations, both ligands in combination promote gene transactivation. Therefore, we first determined the response of the neonatal rat lung to treatments with all-trans-RA, 9-cis-RA, and a 1:1 combination of both isomers, administered in the absence of VA or combined with a supplement of VA (molar ratio of 10 VA:1 acidic retinoid). Treatments were given daily on postnatal days 5, 6, and 7, and lung and plasma retinol were determined on day 8. Fig. 1A shows that RA produced a small increase in lung RE when given alone, as we have shown previously (12Ross A.C. Ambalavanan N. Zolfaghari R. Li N-q. Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in neonatal rats.J. Lipid Res. 2006; 47: 1844-1851Google Scholar), indicating that RA influences the metabolism of endogenous retinol in the absence of VA supplementation. When RA was combined with VA as VARA, lung RE increased ∼7 times higher compared with treatment with VA alone. In contrast to all-trans-RA, 9-cis-RA in the absence of VA had no effect on lung RE, suggesting this isomer does not significantly regulate the metabolism of endogenous retinol in the lungs. However, similar to all-trans-RA, 9-cis-RA produced a significant although slightly lower synergy when given with VA. The combination of at-RA and 9-cis-RA (mixed 1:1, with total RA equal to that given as either isomer alone) had no additional effect. The concentration of plasma retinol (Fig. 1B) was within the normal range for neonates in all treatment groups. Plasma retinol levels were ∼30% higher for each treatment that included VA, regardless of whether an acidic retinoid was coadministered (all P < 0.05), whereas none of the acidic retinoids alone had a significant effect. Therefore, plasma retinol responded only to the VA component of the dose. From this initial study, we confirmed that at-RA is able to provide a significant boost to VA (e.g., as VARA) by increasing lung RE content ∼5–7 times. To further test the interaction of VA and acidic retinoids on lung RE formation and to examine regulatory processes, we next compared all-trans-RA, as the predominant natural regulatory isomer, and a retinobenzoic acid analog of RA, Am580, which has been shown to relatively resistant to metabolism and to bind selectively to the RAR-α form of nuclear retinoid receptors (29Kagechika H. Novel synthetic retinoids and separation of the pleiotropic retinoidal activities.Curr. Med. Chem. 2002; 9: 591-608Google Scholar). All-trans-RA and Am580, with and without VA, were studied for their ability to promote lung RE formation, enhance the uptake of newly absorbed retinol, and alter the expression of genes potentially important for retinol uptake, esterification, and homeostasis. We conducted two studies, one of 6 h and one of 12 h duration, each with groups of 6–7 day old neonates that were treated orally with oil (control), VA alone, RA alone, VARA, Am580 alone, and VAAm. The values for the control groups in the two experiments were very similar; thus, treatment responses for both experiments are presented together for comparison. Lung RE increased after VA supplementation alone, which was evident as early as 6 h after dosing (Fig. 2A). Notably, the magnitude of increase was the same at both 6 and 12 h (Fig. 2B) for the VA, RA, and VARA groups, implying that the uptake of VA and its storage as RE in the lungs is very rapid after oral dosing, reaching a steady state by 6 h. Am580 alone produced a small increase in lung RE at both times (Fig. 2A, B). Thus, Am580, like RA, influences the metabolism of endogenous retinol. However, whereas the synergy with VAAm was significant at both 6 and 12 h, lung RE in the VAAm group was significantly lower at 12 h than at 6 h and lower for the VAAm group than for the VARA group at 12 h. These results indicated that the stable analog Am580 is as effective as RA for promoting RE formation in the very early absorptive period (6 h), while Am580 appeared to have the effect of quickening retinol metablism in the lung, as evidenced by the fall in lung RE in the postabsorptive period, measured at 12 h. In the 12 h study, we included a tracer of [3H]retinol with each treatment to investigate how newly absorbed [3H]retinol is partitioned between the neonatal rat lung and liver, the major storage organ for VA, and whether treatments with VARA and VAAm may promote the uptake of newly absorbed retinol into the lung, which in turn could be a mechanism for the observed increase in lung RE content after VARA or VAAm treatment. The 12 h time point was chosen because we expected intestinal absorption of the [3H]retinol tracer and the VA dose it traced to have been completed by 12 h; thus, the tissue distribution of newly absorbed retinol for the entire dose could be assessed. The percentage of newly absorbed 3H in the lungs did not differ between the VA group and the control group (Fig. 3A), indicating that the supplement of VA alone did not increase the fractional uptake of retinol from the oral dose. However, the uptake of [3H]retinol was significantly higher in the lungs of neonates treated with at-RA, VARA, Am580, and VAAm (all P < 0.05 versus control). From these results, it can be inferred that VA itself did not affect the uptake of newly absorbed retinol, whereas both at-RA and Am580 promoted a higher uptake of newly absorbed retinol, whether or not the mass of VA being absorbed at the time was low, i.e., without VA, or high, after the VA supplement. We noted, however, that more 3H was present in the lungs of noenates treated with VARA group than with VAAm. This result might be due to a difference in the rate of retinol metabolism as affected by at-RA and Am580. We also measured the uptake of the orally administered [3H]retinol into the liver, the major storage organ for VA, for comparison to lung. The percentage of [3H]retinol taken up by the liver was higher, as expected; however, in addition, we noted that the treatment effect in liver differed from that in lung. In the liver, [3H]RE was increased only by VA and VARA (Fig. 3B). We did not expect VARA to increase the mass of liver RE more than VA alone because the VARA synergy has not been observed for liver (12Ross A.C. Ambalavanan N. Zolfaghari R. Li N-q. Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in neonatal rats.J. Lipid Res. 2006; 47: 1844-1851Google Scholar, 17Ross A.C. Li N. Wu L. The components of VARA, a nutrient-metabolite combination of vitamin A and retinoic acid, act efficiently together and separately to increase retinyl esters in the lungs of neonatal rats.J. Nutr. 2006; 136: 2803-2807Google Scholar). Similarly, VAAm did not increase liver RE content more than VA alone. However, liver from the VAAm-treated group did not show the same increase in [3H-retinol as in the VA and the VARA groups. We surmised that this might be due to a higher rate of retinol metabolism in the VAAm group. The difference between VARA and VAAm did not appear to be due to a difference in the fractional conversion of newly absorbed retinol to RE, as the percentage of 3H present as [3H]RE was similar in the VARA and VAAm groups (data not shown). The rapid effect of VARA and VAAm on retinol uptake and RE accumulation in the lungs suggested that if changes in gene expression are responsible, they must occur early after treatment. We thus determined the expression of several genes implicated in retinoid homeostasis in the lungs of neonates in the 6 and 12 h studies. Gene expression was determined by quantitative PCR for STRA6, LRAT, and two members of the CYP26 family, CYP26A1 and CYP26B1, as well as the cellular retinol-binding protein (CRBP1). Results for STRA6, a
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