Multiple cytochrome P-450 genes are concomitantly regulated by vitamin A under steady-state conditions and by retinoic acid during hepatic first-pass metabolism.

Vitamin A (retinol) is an essential precursor for the production of retinoic acid (RA), which in turn is a major regulator of gene expression, affecting cell differentiation throughout the body. Understanding how vitamin A nutritional status, as well as therapeutic retinoid treatment, regulates the expression of retinoid homeostatic genes is important for improvement of dietary recommendations and therapeutic strategies using retinoids. This study investigated genes central to processes of retinoid uptake and storage, release to plasma, and oxidation in the liver of rats under steady-state conditions after different exposures to dietary vitamin A (deficient, marginal, adequate, and supplemented) and acutely after administration of a therapeutic dose of all-trans-RA. Over a very wide range of dietary vitamin A, lecithin:retinol acyltransferase (LRAT) as well as multiple cytochrome P-450s (CYP26A1, CYP26B1, and CYP2C22) differed by diet and were highly correlated with one another and with vitamin A status assessed by liver retinol concentration (all correlations, P < 0.05). After acute treatment with RA, the same genes were rapidly and concomitantly induced, preceding retinoic acid receptor (RAR)ß, a classical direct target of RA. CYP26A1 mRNA exhibited the greatest dynamic range (change of log 2(6) in 3 h). Moreover, CYP26A1 increased more rapidly in the liver of RA-primed rats than naive rats, evidenced by increased CYP26A1 gene expression and increased conversion of [(3)H]RA to polar metabolites. By in situ hybridization, CYP26A1 mRNA was strongly regulated within hepatocytes, closely resembling retinol-binding protein (RBP)4 in location. Overall, whether RA is produced endogenously from retinol or administered exogenously, changes in retinoid homeostatic gene expression simultaneously favor both retinol esterification and RA oxidation, with CYP26A1 exhibiting the greatest dynamic change.

Retinoids synergize with insulin to induce hepatic Gck expression.

Hepatic GK (glucokinase) plays a key role in maintaining glucose homoeostasis. Many stimuli regulate GK activity by controlling its gene transcription. We hypothesized that endogenous lipophilic molecules modulate hepatic Gck expression. Lipophilic molecules were extracted from rat livers, saponified and re-constituted as an LE (lipophilic extract). LE synergized with insulin to induce primary hepatocyte, but not beta-cell, Gck expression in an SREBP-1c (sterol-regulatory-element-binding protein-1c)-independent manner. The dramatic induction of Gck mRNA resulted in a significant increase in GK activity. Subsequently, the active molecules were identified as retinol and retinal by MS after the purification of the active LE fractions. Retinoids synergized with insulin to induce Gck expression by the activation of both RAR [RA (retinoic acid) receptor] and RXR (retinoid X receptor). Inhibition of RAR activation completely abolished the effect of retinal. The hepatic GK specific activity and Gck mRNA levels of Zucker lean rats fed with a VAD [VA (vitamin A)-deficient] diet were significantly lower than those of rats fed with VAS (VA-sufficient) diet. Additionally, the hepatic Gck mRNA expression of Sprague-Dawley rats fed with a VAD diet was lower than that of rats fed with VA-marginal, -adequate or -supplemented diets. The reduced expression of Gck mRNA was increased after an intraperitoneal dose of RA in VAD rats. Furthermore, an intravenous injection of RA rapidly raised hepatic Gck expression in rats fed with a VAS control diet. Understanding the underlying mechanism that mediates the synergy may be helpful for developing a treatment strategy for patients with diabetes.

Retinol combined with retinoic acid increases retinol uptake and esterification in the lungs of young adult rats when delivered by the intramuscular as well as oral routes.

The lungs require an adequate supply of vitamin A for normal embryonic development, postnatal maturation, and maintenance and repair during adult life. We have previously shown that a nutrient-metabolite combination of vitamin A admixed with a small proportion (10%) of retinoic acid (RA), referred to as VARA, acts synergistically to increase lung retinyl ester (RE) concentration in neonatal rats. A series of studies was designed to test whether VARA increases RE in adult lungs, and whether VARA is more effective than vitamin A when given by the i.m. route. Orally administered VARA increased RE in the lungs of vitamin A-marginal adult rats more than either vitamin A or RA alone (P < 0.05). In vitamin A-deficient young adult rats, lung RE was increased by VARA when administered by the i.m. route. When a tracer of (3)H-retinol was added to the placebo (oil), vitamin A, and VARA doses, total (3)H and (3)H-RE increased in the lungs more with VARA than vitamin A alone, for oral and i.m. dosing. Nevertheless, when VARA and vitamin A were given by the oral route, they were more effective in increasing RE in the liver. Plasma retinol was increased similarly in vitamin A-deficient rats after administration of VARA and vitamin A, by either the oral or the i.m. route. Overall, VARA can increase retinol uptake and esterification in adult lungs when delivered intramuscularly as well as orally.

Lung retinyl ester is low in young adult rats fed a vitamin A deficient diet after weaning, despite neonatal vitamin A supplementation and maintenance of normal plasma retinol.

Although it is understood that plasma retinol concentration is not proportional to the concentration of vitamin A stored in liver, plasma retinol still is often used as an indicator of vitamin A status. An aim of vitamin A supplementation strategies is to maintain plasma retinol concentration in a range considered adequate, generally >1.05 micromol/L in humans, with some adjustment for age. In the present study in rats, we addressed the following question: Does lung vitamin A increase postnatally, as is observed in rats fed a vitamin A-adequate diet, if plasma retinol is maintained at approximately 1 micromol/L by supplementation at neonatal age, but the weaning diet is deficient in vitamin A? We treated rats on postnatal d 6, 7, and 8 with placebo (oil), vitamin A, retinoic acid (RA), and a nutrient-metabolite combination of vitamin A and RA, VARA, after which tissues were analyzed on d 9. Other rats treated identically as neonates were fed a vitamin A-deficient diet from 3-9 wk of age, and in parallel, another group of rats was fed a vitamin A-adequate diet. Although supplementation with vitamin A or VARA elevated liver and lung retinyl esters (RE) on d 9 (P < 0.0001), and prevented the fall in plasma retinol to <1 micromol/L by 9 wk of age, when the diet was vitamin A-deficient, lung RE fell to 28% of the concentration present in the lungs of rats fed the vitamin A-adequate diet (P < 0.0001). We infer that the lungs depend, at least in part, on the uptake of dietary vitamin A, probably from chylomicrons, to develop RE stores in the postweaning growth period.

Neonatal-age treatment with vitamin A delays postweaning vitamin A deficiency and increases the antibody response to T-cell dependent antigens in young adult rats fed a vitamin A-deficient diet.

Vitamin A supplementation for infants and young children is recommended by WHO/UNICEF for countries with a high prevalence of vitamin A deficiency, and vitamin A is often administered at immunization contacts. Using a rat model, we tested whether supplementation with vitamin A or other retinoids at the time of neonatal immunization has prospective benefit in terms of preventing postweaning vitamin A deficiency and promoting antibody responses to T-cell dependent (TD) antigens administered at the neonatal stage and at the young adult stage. Rats were treated orally on postnatal d 6-8 with oil (placebo control), vitamin A, retinoic acid, or a combination of both (VARA) (n > or = 12/group), and immunized with tetanus toxoid (TT) on d 7. The primary anti-TT response was measured on d 21, after which weanling rats were fed the vitamin A-deficient diet until approximately 10 wk. At 8 wk, rats were immunized again with TT to determine the recall response, and with a novel TD antigen, keyhole limpet hemocyanin (KLH), to assess the adult primary response. None of the supplements affected the plasma titer of anti-TT immunoglobulin G (IgG) on d 21 (P = 0.25). However, neonatal-age supplementation with vitamin A or VARA at the young adult stage resulted in: >5 times higher anti-TT IgG recall response (P < 0.01); 5- and 9-times higher anti-KLH primary IgM and IgG responses, respectively (P < 0.05), and plasma retinol in the normal range (approximately 1.0 micromol/L vs. approximately 0.35 micromol/L in retinoic acid-treated and control groups, P < 0.0001). We conclude that early-life supplementation with vitamin A or VARA can prospectively benefit the primary and recall antibody responses to TD antigens administered at the young adult stage, which may involve the maintenance of normal plasma retinol levels.

Lipopolysaccharide opposes the induction of CYP26A1 and CYP26B1 gene expression by retinoic acid in the rat liver in vivo.

Retinoic acid (RA), a principal metabolite of vitamin A (retinol), is an essential endogenous regulator of gene transcription and an important therapeutic agent. The catabolism of RA must be well regulated to maintain physiological concentrations of RA. The cytochrome P450 (CYP) gene family CYP26, which encodes RA-4-hydroxylase activity, is strongly implicated in the oxidation of RA. Inflammation alters the expression of numerous genes; however, whether inflammation affects CYP26 expression is not well understood. We investigated the regulation of CYP26A1 and CYP26B1 mRNA levels by RA and LPS in the rat liver, as the liver is centrally involved in retinoid metabolism and the acute-phase response to LPS. Both CYP26A1 and CYP26B1 mRNA were induced in <4 h by a single oral dose of all-trans-RA. RA-induced responses of both CYP26A1 and CYP26B1 were significantly attenuated in rats with LPS-induced inflammation whether LPS was given concurrently with RA or after the RA-induced increase in CYP26A1 and CYP26B1 mRNA levels. When RA and LPS were administered simultaneously (6-h study), LPS alone had little effect on either CYP26A1 or CP26B1 mRNA, but LPS reduced by 80% the RA-induced increase in CYP26A1 mRNA (P<0.02), with a similar trend for CYP26B1 mRNA. When LPS was administered 4 h after RA (16-h study), it abrogated the induction of CYP26A1 (P<0.02) and CYP26B1 (P<0.01). Overall, these results suggest that inflammation can potentially disrupt the balance of RA metabolism and maintenance of RA homeostasis, which may possibly affect the expression of other RA-regulated genes.

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.

Retinoic acid (RA), produced from vitamin A (VA, retinol), is required for normal lung development and postnatal lung maturation. The concentration of retinyl ester (RE), the major storage form of retinol, decreases in the lungs in the perinatal period. Previously, we tested VARA, a nutrient-metabolite combination of VA and RA (10:1 molar ratio), on lung RE formation in postnatal rats and showed that the components of VARA acted synergistically to increase lung RE, as compared with the effects of equal amounts of VA and RA given alone. In this study, we first determined the equivalency of orally administered VARA in comparison to a standard oral supplement of VA, with respect to lung and liver RE storage. In a dose-dilution study, VARA was 4 times as effective as the standard dose of VA (VARA-25% did not differ from VA-100%). The synergistic effect of VARA was selective for the lungs, compared with the liver, in which VA and VARA had equal effects. Secondly, we tested whether the 2 components of VARA must be coadministered to exert their synergistic effect on lung RE content. RA and VA and were administered separately and together as VARA. Although RA alone had no effect on lung RE in this 24-h experiment, RA synergized with VA administered either 12 h before RA or 12 h after RA, as well as when coadministered as VARA. We infer that VA and RA are both limiting for lung RE formation in neonates. Given the importance of bioactive retinoids in cell differentiation and lung development, assuring an adequate lung RE content postnatally could be of benefit for lung maturation.

Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in a synergistic manner in neonatal rats.

Vitamin A (VA) is stored in tissues predominantly as retinyl esters (REs), which provide substrate for the production of bioactive retinoids. Retinoic acid (RA), a principal metabolite, has been shown to induce postnatal lung development. To better understand lung RE storage, we compared VA (given as retinyl palmitate), RA, and a nutrient-metabolite combination, VARA, given orally on postnatal days 5-7, for their ability to increase lung RE in neonatal rats. VARA increased lung RE significantly [ approximately 14, 2.4, 2.1, and <1 nmol/g for VARA, VA, RA, and control (C), respectively; P < 0.001]; the increase by VARA was more than additive compared with the effects of VA and RA alone. Lung histology and morphometry were unchanged. In a 6 h metabolic study, providing [(3)H]retinol with VARA, compared with VA or C, increased the uptake of newly absorbed (3)H by 3-fold, indicating that VARA stimulated the uptake of [(3)H]retinol and its retention as [(3)H]RE in neonatal lungs. After cessation of VARA, lung RE remained increased for 9 d afterward, through the period of alveolar development. In conclusion, VARA, a 10:1 nutrient-metabolite combination, increased lung RE significantly compared with VA alone and could be a promising therapeutic option for enhancing the delivery of VA to the lungs.