Retinoid resistance and multifaceted impairment of retinoic acid synthesis in glioblastoma.

Measuring concentrations of the differentiation-promoting hormone retinoic acid (RA) in glioblastoma tissues would help to understand the reason why RA treatment has been inefficient in clinical trials involving brain tumor patients. Here, we apply a recently established extraction and measurement protocol to screen glioblastoma tissues for the levels of the RA precursor retinol and biologically active RA. Combining this approach with mRNA analyses of 26 tumors and 8 normal brains, we identify a multifaceted disturbance of RA synthesis in glioblastoma, involving multiple aldehyde dehydrogenase 1 family and retinol dehydrogenase enzymes. Through database studies and methylation analyses, we narrow down chromosomal deletions and aberrant promoter hypermethylation as potential mechanisms accounting for these alterations. Employing chromatin immunoprecipitation analyses and cell-culture studies, we further show that chromatin at RA target genes is poised to RA substitution, but most glioblastoma cell cultures are completely resistant to RA treatment. This paradoxical RA response is unrelated to alternative RA signaling through the fatty acid-binding protein 5/peroxisome proliferator-activated receptor delta axis. Our data suggest a multifaceted disturbance of RA synthesis in glioblastoma and contribute to reconsider current RA treatment strategies.

LRAT-specific domain facilitates vitamin A metabolism by domain swapping in HRASLS3.

Cellular uptake of vitamin A, production of visual chromophore and triglyceride homeostasis in adipocytes depend on two representatives of the vertebrate N1pC/P60 protein family, lecithin:retinol acyltransferase (LRAT) and HRAS-like tumor suppressor 3 (HRASLS3). Both proteins function as lipid-metabolizing enzymes but differ in their substrate preferences and dominant catalytic activity. The mechanism of this catalytic diversity is not understood. Here, by using a gain-of-function approach, we identified a specific sequence responsible for the substrate specificity of N1pC/P60 proteins. A 2.2-Å crystal structure of the HRASLS3-LRAT chimeric enzyme in a thioester catalytic intermediate state revealed a major structural rearrangement accompanied by three-dimensional domain swapping dimerization not observed in native HRASLS proteins. Structural changes affecting the active site environment contributed to slower hydrolysis of the catalytic intermediate, supporting efficient acyl transfer. These findings reveal structural adaptation that facilitates selective catalysis and mechanism responsible for diverse substrate specificity within the LRAT-like enzyme family.

Identification of the 11-cis-specific retinyl-ester synthase in retinal Müller cells as multifunctional O-acyltransferase (MFAT).

Absorption of a photon by a rhodopsin or cone-opsin pigment isomerizes its 11-cis-retinaldehyde (11-cis-RAL) chromophore to all-trans-retinaldehyde (all-trans-RAL), which dissociates after a brief period of activation. Light sensitivity is restored to the resulting apo-opsin when it recombines with another 11-cis-RAL. Conversion of all-trans-RAL to 11-cis-RAL is carried out by an enzyme pathway called the visual cycle in cells of the retinal pigment epithelium. A second visual cycle is present in Müller cells of the retina. The retinol isomerase for this noncanonical pathway is dihydroceramide desaturase (DES1), which catalyzes equilibrium isomerization of retinol. Because 11-cis-retinol (11-cis-ROL) constitutes only a small fraction of total retinols in an equilibrium mixture, a subsequent step involving selective removal of 11-cis-ROL is required to drive synthesis of 11-cis-retinoids for production of visual chromophore. Selective esterification of 11-cis-ROL is one possibility. Crude homogenates of chicken retinas rapidly convert all-trans-ROL to 11-cis-retinyl esters (11-cis-REs) with minimal formation of other retinyl-ester isomers. This enzymatic activity implies the existence of an 11-cis-specific retinyl-ester synthase in Müller cells. Here, we evaluated multifunctional O-acyltransferase (MFAT) as a candidate for this 11-cis-RE-synthase. MFAT exhibited much higher catalytic efficiency as a synthase of 11-cis-REs versus other retinyl-ester isomers. Further, we show that MFAT is expressed in Müller cells. Finally, homogenates of cells coexpressing DES1 and MFAT catalyzed the conversion of all-trans-ROL to 11-cis-RP, similar to what we observed with chicken-retina homogenates. MFAT is therefore an excellent candidate for the retinyl-ester synthase that cooperates with DES1 to drive synthesis of 11-cis-retinoids by mass action.

Genetic dissection of retinoid esterification and accumulation in the liver and adipose tissue.

Approximately 80-90% of all retinoids in the body are stored as retinyl esters (REs) in the liver. Adipose tissue also contributes significantly to RE storage. The present studies, employing genetic and nutritional interventions, explored factors that are responsible for regulating RE accumulation in the liver and adipose tissue and how these influence levels of retinoic acid (RA) and RA-responsive gene expression. Our data establish that acyl-CoA:retinol acyltransferase (ARAT) activity is not involved in RE synthesis in the liver, even when mice are nutritionally stressed by feeding a 25-fold excess retinol diet or upon ablation of cellular retinol-binding protein type I (CRBPI), which is proposed to limit retinol availability to ARATs. Unlike the liver, where lecithin:retinol acyltransferase (LRAT) is responsible for all RE synthesis, this is not true for adipose tissue where Lrat-deficient mice display significantly elevated RE concentrations. However, when CrbpI is also absent, RE levels resemble wild-type levels, suggesting a role for CrbpI in RE accumulation in adipose tissue. Although expression of several RA-responsive genes is elevated in Lrat-deficient liver, employing a sensitive liquid chromatography tandem mass spectrometry protocol and contrary to what has been assumed for many years, we did not detect elevated concentrations of all-trans-RA. The elevated RA-responsive gene expression was associated with elevated hepatic triglyceride levels and decreased expression of Pparδ and its downstream Pdk4 target, suggesting a role for RA in these processes in vivo.

GS2 as a retinol transacylase and as a catalytic dyad independent regulator of retinylester accretion.

GS2 (PNPLA4; iPLAeta) is the smallest member of the patatin-like family of phospholipases (PNPLA). It was initially identified by its ability to hydrolyze retinylesters (RE) in cell homogenates, and was later found to esterify retinol using a variety of acyl donors. In the present study we set out to determine its cellular function and examined its impact on RE status in 293T cells transfected with GS2, GS2-M1 (a non-translatable mutant of GS2) and empty vector, in fibroblasts isolated from normal and GS2-null donors and in SCC12b and in a somatic cell knock-out of GS2 (SCC12b-GS2(neo/-)), that we generated by homologous recombination. At 50nM medium retinol, GS2 had no significant impact on RE accumulation. However, at 2muM retinol, GS2 promoted a 1.6- to 5-fold increase in RE accumulation. To verify role of GS2 as a catalyst, RE levels were measured in 293T transfected wild type GS2, catalytic dyad mutants devoid of enzymatic activity, or alanine substitution mutants spanning the entire GS2 sequence. Surprisingly, every GS2 mutant promoted RE accumulation. This activity was also observed in the GS2 paralogues and rat orthologue. The data demonstrate that within the context of the cell GS2 promotes RE accumulation and may do so either as a catalyst or as a regulatory protein that enhances RE formation catalyzed by other acyl transferases.

Retinol Esterification by DGAT1 Is Essential for Retinoid Homeostasis in Murine Skin.

Retinoic acid (RA) is a potent signaling molecule that is essential for many biological processes, and its levels are tightly regulated by mechanisms that are only partially understood. The synthesis of RA from its precursor retinol (vitamin A) is an important regulatory mechanism. Therefore, the esterification of retinol with fatty acyl moieties to generate retinyl esters, the main storage form of retinol, may also regulate RA levels. Here we show that the neutral lipid synthesis enzyme acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) functions as the major acyl-CoA:retinol acyltransferase (ARAT) in murine skin. When dietary retinol is abundant, DGAT1 deficiency results in elevated levels of RA in skin and cyclical hair loss; both are prevented by dietary retinol deprivation. Further, DGAT1-deficient skin exhibits enhanced sensitivity to topically administered retinol. Deletion of the enzyme specifically in the epidermis causes alopecia, indicating that the regulation of RA homeostasis by DGAT1 is autonomous in the epidermis. These findings show that DGAT1 functions as an ARAT in the skin, where it acts to maintain retinoid homeostasis and prevent retinoid toxicity. Our findings may have implications for human skin or hair disorders treated with agents that modulate RA signaling.

Retinoid processing in cone and Müller cell lines.

To determine whether cones and Müller cells in the rod dominated retina cooperate to regenerate the 11-cis retinal chromophore via the retinoid cycle, two cell lines from the rod dominated retinas of Murine were used for this study: 661W, a mouse cell line derived from cones, and rMC-1, a rat Müller cell line. Retinoid cycle enzymes were analyzed by RT-PCR, and their catalytic activity was detected by incubation with retinoids and analyzed by HPLC. We found that 661W cells are capable of reducing all-trans retinal to all-trans retinol due to the presence of multiple dehydrogenases and to generate minor amounts of retinyl-ester. The rMC-1 cells take up all-trans retinol and oxidize it to all-trans retinal or esterify it to retinyl-ester, but are incapable of isomerizing all-trans retinoids to 11-cis retinoids. This could be a reflection of lack of necessary activities in Müller cells in vivo, which suggests that Müller cells do not contribute to retinoid cycling by regenerating 11-cis retinoids. Alternatively, this could be due to the potential that rMC-1, as a transformed cell line, has stopped expressing the proteins needed for the regeneration of 11-cis retinoids.

11-cis-Acyl-CoA:retinol O-acyltransferase activity in the primary culture of chicken Muller cells.

A novel retinoid cycle has recently been identified in the cone-dominated chicken retina, and this cone cycle accumulates 11-cis-retinyl esters upon light adaptation. The purpose of this study is to investigate how 11-cis-retinyl esters are formed in the retina. Primary cultures of chicken Muller cells and cell membrane were incubated with all-trans- or 11-cis-retinol to study retinyl ester synthesis. In Muller cells, esterification of 11-cis-retinol was four times greater than esterification of all-trans-retinol. In the presence of palmitoyl-CoA and CRALBP, Muller cell membranes synthesized 11-cis-retinyl ester from 11-cis-retinol at a rate which was 20-fold higher than that of all-trans-retinyl ester. In the absence of CRALBP, 11-cis-retinyl ester synthesis was greatly reduced (by 7-fold). In the absence of palmitoyl-CoA, retinyl ester synthesis was not observed. Muller cell membranes incubated with radiolabeled palmitoyl-CoA resulted in the transfer of the labeled acyl group to retinol. This acyl transfer was greatly reduced in the presence of progesterone, a known ARAT inhibitor. 11-cis-ARAT activity remained unchanged when assayed in the presence of all-trans-retinol, suggesting a distinct catalytic activity from that of all-trans-ARAT. Apparent kinetic rates for 11-cis-ARAT were 0.135 nmol min(-)(1) mg(-)(1) (V(max)) and 11.25 microM (K(M)) and for all-trans-ARAT were 0.0065 nmol min(-)(1) mg(-)(1) (V(max)) and 28.88 microM (K(M)). Our data indicate that Muller cells in the chicken retina possess 11-cis-ARAT activity, thus providing an explanation for the accumulation of 11-cis-retinyl esters in the cone cycle.

Acyl CoA:retinol acyltransferase (ARAT) activity is present in bovine retinal pigment epithelium.

Visual perception is mediated by a family of G protein-coupled receptors called the opsins. The light-absorbing chromophore in most opsins is 11-cis-retinaldehyde, which is isomerized to all-trans-retinaldehyde upon absorption of a photon. Restoration of light sensitivity to the photobleached opsin requires chemical re-isomerization of the chromophore. This is carried out by an enzymatic pathway called the visual cycle in retinal pigment epithelial cells. The isomerase in this pathway uses fatty-acyl esters of all-trans-retinol as substrate. A retinyl-ester synthase that produces these esters, called lecithin:retinol acyltransferase (LRAT), has been extensively characterized. Based on prior biochemical studies and the phenotype in lrat(-/-) knockout mice, it has been assumed that LRAT is the sole or dominant retinyl-ester synthase in the retinal pigment epithelium. Here we demonstrate the presence of a second ester synthase activity in these cells called acyl CoA:retinol acyltransferase (ARAT). We show that this activity uses palmitoyl coenzyme A as an acyl donor, unlike LRAT which uses phosphatidylcholine. Similar to LRAT, ARAT esterifies both all-trans-retinol and 11-cis-retinol. LRAT and ARAT are both potently inhibited by the retinyl-ester analog, all-trans-retinylbromoacetate, but only ARAT is inhibited by progesterone. Unexpectedly, the maximum turnover rate (V(max)) of ARAT was similar to that of LRAT. However, the Michaelis constant (K(M)) of ARAT was 10-fold higher than the K(M) of LRAT for all-trans-retinol. These observations suggest that ARAT may complement LRAT to provide additional retinyl-ester synthase activity under conditions of high all-trans-retinol. These conditions occur in the retina following exposure to bright light.

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A: diacylglycerol acyltransferase 1.

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K(m) values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9+/-2.1 microM and 13.9+/-0.3 microM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits approximately 85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl-oleate formation without influencing the retinyl-palmitate formation. Using this inhibitor, we estimate that approximately 64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.

A human skin multifunctional O-acyltransferase that catalyzes the synthesis of acylglycerols, waxes, and retinyl esters.

Acyl-CoA-dependent O-acyltransferases catalyze reactions in which fatty acyl-CoAs are joined to acyl acceptors containing free hydroxyl groups to produce neutral lipids. In this report, we characterize a human multifunctional O-acyltransferase (designated MFAT) that belongs to the acyl-CoA:diacylglycerol acyltransferase 2/acyl-CoA:monoacylglycerol acyltransferase (MGAT) gene family and is highly expressed in the skin. Membranes of insect cells and homogenates of mammalian cells overexpressing MFAT exhibited significantly increased MGAT, acyl-CoA:fatty acyl alcohol acyltransferase (wax synthase), and acyl-CoA:retinol acyltransferase (ARAT) activities, which catalyze the synthesis of diacylglycerols, wax monoesters, and retinyl esters, respectively. Furthermore, when provided with the appropriate substrates, intact mammalian cells overexpressing MFAT accumulated more waxes and retinyl esters than control cells. We conclude that MFAT is a multifunctional acyltransferase that likely plays an important role in lipid metabolism in human skin.

Molecular logic of 11-cis-retinoid biosynthesis in a cone-dominated species.

The biochemical pathway to visual chromophore biosynthesis in rod-dominated animals involves minimally a two component system in which all-trans-retinyl esters, generated by the action of lecithin retinol acyltransferase (LRAT) on vitamin A, are processed into 11-cis-retinol by isomerohydrolase. Possible differences in retinoid metabolism in cone-dominated animals have been noted in the literature, so it was of interest to explore whether these differences are tangential or fundamental. Central to this issue is whether cone-dominated animals use an isomerohydrolase (IMH)-based mechanism in the predominant pathway to 11-cis-retinoids. Here, it is shown that all-trans-retinyl esters (tREs) are the direct precursors of 11-cis-retinol formation in chicken retinyl pigment epithelium/retina preparations. This conclusion is based on at least three avenues of evidence. First, reagents that block tRE synthesis from vitamin A also block 11-cis-retinol synthesis. Second, pulse-chase experiments also establish that tREs are the precursors to 11-cis-retinol. Finally, 11-cis-retinyl-bromoacetate, a known affinity-labeling agent of isomerohydrolase, also blocks chromophore biosynthesis in the cone system.

Topical delivery of retinoids counteracts the UVB-induced epidermal vitamin A depletion in hairless mouse.

UVB irradiation depletes all-trans-retinol (ROL) and all-trans-retinyl esters (RE) from the hairless mouse epidermis. Prevention of this may be of relevance in counter-acting the long-term side effects of UVB exposure. We studied the effects of a topical treatment with natural retinoids before and after UVB exposure on three parameters involved in vitamin A metabolism: the amount of epidermal ROL and RE, the level of functional cellular retinol-binding protein I (CRBP-I), which is likely to protect ROL from UVB, as well as the cytosolic and microsomal enzyme activities which generate ROL and RE, i.e. all-trans-retinaldehyde (RAL) reductase, acylCoA:retinol acyltransferase (ARAT) and retinyl-ester hydrolase (REH). Topical pretreatment with retinoids promoted a dramatic increase of epidermal ROL, RE and CRBP-I levels, a transient increase of RAL reductase and ARAT activities as well as a decreased activity of REH, indicating a direction of epidermal vitamin A metabolism toward storage. In untreated mice UVB irradiation induced a depletion of epidermal ROL and RE in 10 min and a 50% decrease of CRBP-I after 24 h. In mice treated with topical retinoids, and then exposed to UVB, epidermal RE levels were higher than in vehicle-treated, nonirradiated mice. In contrast, ROL was as much depleted after UVB in pretreated as in untreated animals in spite of an induction of CRBP-I, indicating that CRBP-I does not actually protect ROL from UVB-induced depletion in this model. However, the reconstitution of both epidermal ROL and RE, after their depletion induced by UVB, was accelerated by previous topical treatment with RAL. Our results indicate that topical delivery of retinoids partly counteracts UVB-induced vitamin A depletion and promotes recovery.

Coordinated distribution patterns of three enzyme activities involved in the absorption and metabolism of beta-carotene and vitamin A along the villus-crypt axis of chick duodenum.

The conversion of beta-carotene to retinal and the succeeding metabolic process of the retinal leading to production of retinol and retinyl esters are the prerequisite for the utilization of beta-carotene as a provitamin A. These processes are participated by beta-carotene cleavage enzyme, retinal reductase and retinol esterifying enzyme(s) in the small intestine. To examine whether these enzymes exhibit the coordinated distribution in the villus, we have used the cryostat sectioning technique to quantify the activities of beta-carotene cleavage enzyme, retinal reductase and retinol esterifying enzymes along the villus-crypt axis in 8-day-old chick duodenum. The beta-carotene cleavage enzyme activity was very low in the crypt and gradually increased, reaching a maximum in the mid-villus. The villus-crypt gradient of the beta-carotene cleavage enzyme activity corresponded with those of retinal reductase activity and lecithin: retinol acyltransferase (LRAT) activity, but distinct from that of acyl-CoA: retinol acyltransferase (ARAT) activity. Furthermore, the distribution of the content of retinyl esters was similar to that of LRAT activity. These results suggest that the beta-carotene cleavage enzyme is coordinately distributed along the villus-crypt axis with retinal reductase and LRAT, the two enzymes which require cellular retinol-binding protein, typeII (CRBPII) as the donor of the substrate.

Developmental induction and villus-crypt distribution of retinol esterifying enzyme activities in chick duodenum.

Retinol absorbed and generated from dietary beta-carotene can be esterified by retinol esterifying enzyme(s) in intestinal absorptive cells. In this study, we observed the developmental changes and villus-crypt distribution of the activities of two retinol esterifying enzymes (lecithin-retinol acyltransferase (LRAT); and acyl-CoA-retinol acyltransferase (ARAT) in chick duodenum) to seek the possibility that these enzymes play distinct roles in retinol absorption and metabolism. Intestinal LRAT activity was barely expressed in embryonic stages until 2-3 d before hatching, when its activity becomes detectable; thereafter it abruptly increased to the maximal level at the third day of the posthatch period. In contrast, ARAT activity was present in the duodenum at the earliest stage examined, the 15th day of embryogenesis, and was elevated to the maximal level 3-4 d after hatching. An assay of LRAT and ARAT activities along the villus-crypt axis of the duodenum by a cryostat sectioning technique revealed that between the day of hatching and 1 d posthatch, an abrupt induction of LRAT activity occurred only in the villus region of the duodenum, where a coordinated induction of cellular retinol-binding protein, type II (CRBPII), was observed. In contrast, the rise in ARAT activity observed around the hatching period occurred at the broader portions of the villi including the area of villus-crypt junction. These observations in the developmental changes and distribution of LRAT and ARAT activities suggest that LRAT activity but not ARAT activity is closely related to the induction of CRBPII in the duodenum of developing chicks.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the lymphatic absorption of a single oral dose of [3H]retinol and on the intestinal retinol esterification in the rat.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters the turnover of vitamin A in the body and inhibits the normal hepatic accumulation of dietary vitamin A. Vitamin A is absorbed in the small intestine, where it is incorporated into chylomicrons as retinyl esters for release into the lymph and further distributed via blood to the liver for storage. The aim of the present study was to investigate if the decreased hepatic vitamin A levels in TCDD-exposed rats could be due to impaired intestinal absorption of vitamin A via lymph. Male Sprague-Dawley rats were given a single oral dose of TCDD (10 microg/kg). Five days after administration, the main intestinal lymph duct of the rats was cannulated. After a 24-h recovery from surgery, the rats were each given a single dose of [3H]retinol in corn oil via gavage and the lymph was collected for 24 h. The cumulative radiolabel recovered in the intestinal lymph was significantly lower in TCDD-treated than in control rats during the first 6 h of absorption. However, no significant differences in radiolabel recovered in lymph were seen when looking at the entire 24-h collection period. In the intestinal mucosa, retinol esterification catalyzed by the enzyme lecithin:retinol acyl transferase (LRAT) or acyl coenzyme A (CoA):retinol transferase (ARAT) was not statistically different between the groups. However, mucosal retinyl palmitate levels were significantly increased in TCDD-treated rats. In conclusion, a small and transient reduction was found of the uptake of vitamin A into the lymph of TCDD-treated rats. It is obvious that this finding cannot explain the TCDD-induced decrease in hepatic vitamin A levels in the rat. Rather, a combination of inhibited retinol esterification in hepatic stellate cells, increased release of endogenous vitamin A, and increased hepatic catabolism of retinoids could explain the effect of TCDD on liver retinoid levels.

Radiation inactivation analysis of acyl-CoA:retinol acyltransferase and lecithin:retinol acyltransferase in rat liver.

Microsomes from liver and several other tissues esterify retinol through both fatty acyl-CoA-dependent and -independent reactions. Two activities, acyl-CoA:retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT) activities, have been characterized enzymatically but neither has yet been purified and characterized biochemically. We have used the method of radiation inactivation to determine the target sizes of ARAT and LRAT in intact microsomal membranes from rat liver. After exposure of frozen liver microsomes to ionizing radiation, the activity of ARAT decayed exponentially yielding a target size of 73 +/- 18 kDa (mean +/- SD, n = 6). The activity of LRAT was assayed both by monitoring the esterification of retinol bound to the cellular retinol-binding protein (CRBP) and of solvent-dispersed retinol. With both assays a single exponential was observed with radiation doses of 9 to 150 Mrads. The slopes obtained with both LRAT assays were similar, yielding target sizes of 52 +/- 10 kDa (n = 10) for the LRAT assay with CRBP-retinol and 56 +/- 7 kDa (n = 6) for the LRAT assay with dispersed retinol. These target sizes did not differ from each other but were significantly smaller than that of ARAT. These data provide the first physical evidence of the independent entities which catalyze the ARAT and LRAT reactions of liver microsomes.

Vitamin A status regulates hepatic lecithin: retinol acyltransferase activity in rats.

The activity of lecithin:retinol acyltransferase (LRAT) was determined in microsomes from the liver and small intestine of rats with differing vitamin A status. In animals depleted of retinol, as judged by undetectable liver vitamin A stores and low plasma retinol concentrations, hepatic LRAT activity was almost undetectable, whether assayed with retinol bound to cellular retinol-binding protein or solvent-dispersed retinol. In contrast, neither the activity of intestinal LRAT nor that of acyl-CoA:retinol acyltransferase in either liver or intestine differed from that of vitamin A-adequate rats. During the course of vitamin A depletion, liver LRAT activity fell progressively, nearly in parallel to the decrease in plasma retinol concentration. Oral repletion of vitamin A-depleted rats with 0.8 mg of retinol resulted in a very rapid restoration of plasma retinol concentration and full recovery of hepatic LRAT activity within 24 h, together with deposition of retinyl ester in the liver. These data strongly implicate LRAT activity in liver as responsible for the storage of hepatic retinyl esters. Retention of the intestine's capacity to esterify retinol during vitamin A deficiency provides a mechanism for capture of dietary vitamin A, while reduced hepatic LRAT activity may function to redirect retinol in liver from storage to other metabolic pathways.

Age-related effects of chronic ethanol intake on vitamin A status in Fisher 344 rats.

The present study was designed to investigate the interaction of age and ethanol on vitamin A status in rats. Rats aged 2 and 19 mo were fed a liquid diet containing 36% of total energy as ethanol or pair-fed a diet containing isoenergetic carbohydrate in place of ethanol. After 3 wk older rats had lower serum retinol (P = 0.04) and higher vitamin A concentrations in liver (P = 0.0001), esophagus (P = 0.0001) and the proximal (P = 0.03) and distal (P = 0.0001) colon than younger animals. Hepatic microsomal cytochrome P-450, retinyl ester hydrolase (REH) and cellular retinol-binding protein (cRBP) were significantly reduced; acyl coenzyme A: retinol acyltransferase (ARAT) was increased; and alcohol (retinol) dehydrogenase (ADH) activity was unchanged with age. Ethanol ingestion increased serum retinol as well as esophageal and colonic vitamin A levels in both age groups. Hepatic cRBP decreased further in the older rats with ethanol feeding, but no change was noted in the percentage of hepatic vitamin A as retinol or retinyl esters. Ethanol ingestion decreased REH (P = 0.0001) and ARAT activities (P = 0.02) and increased cytochrome P-450 (P = 0.04) but had no effect on the activity of ADH in either age group. These data indicate that, regardless of age, chronic ethanol ingestion significantly alters the tissue distribution of vitamin A; however, ethanol reduced cRBP levels only in older rats.