Retinol Acyltransferase Activity Research Articles

Characterization of an Acyl‐CoA:retinol Acyltransferase (ARAT) Activity in the Hepatopancreas of the American Lobster (Homarus americanus)

Vitamin A (retinol) is a necessary nutrient in higher animals. In mammals, the pathways by which retinol is transported and metabolized are well described. One important reaction in these pathways is the conversion of retinol into retinyl esters, which occurs in several tissues, including the small intestine, liver, and eye. Little is known about the conversion of retinol into retinyl esters in invertebrates. We have identified an acyl‐CoA:retinol acyltransferase (ARAT) activity in the hepatopancreas of the American lobster (Homarus americanus). This activity shows a linear dependence on time and microsome concentration. It shows temperature dependence and an optimal pH of 6.5. We have characterized its acyl‐CoA preference and determined Km and Vmax values for both retinol and acyl‐CoA.Support or Funding InformationCentre College

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.

β‐Carotene and its cleavage enzyme β‐carotene‐15,15′‐oxygenase (CMOI) affect retinoid metabolism in developing tissues

The mammalian embryo relies on maternal circulating retinoids (vitamin A derivatives) for development. β-Carotene is the major human dietary provitamin A. β-Carotene-15,15'-oxygenase (CMOI) has been proposed as the main enzyme generating retinoid from β-carotene in vivo. CMOI is expressed in embryonic tissues, suggesting that β-carotene provides retinoids locally during development. We performed loss of CMOI function studies in mice lacking retinol-binding protein (RBP), an established model of embryonic vitamin A deficiency (VAD). We show that, unexpectedly, lack of CMOI in the developing tissues further exacerbates the severity of VAD and thus the embryonic malformations of RBP(-/-) mice. Since β-carotene was not present in any of the mouse diets, we unveiled a novel action of CMOI independent from its β-carotene cleavage activity. We also show for the first time that CMOI exerts an additional function on retinoid metabolism by influencing retinyl ester formation via modulation of lecithin:retinol acyltransferase (LRAT) activity, at least in developing tissues. Finally, we demonstrate unequivocally that β-carotene can serve as an alternative vitamin A source for the in situ synthesis of retinoids in developing tissues by the action of CMOI.

Metabolic Basis of Visual Cycle Inhibition by Retinoid and Nonretinoid Compounds in the Vertebrate Retina

In vertebrate retinal photoreceptors, the absorption of light by rhodopsin leads to photoisomerization of 11-cis-retinal to its all-trans isomer. To sustain vision, a metabolic system evolved that recycles all-trans-retinal back to 11-cis-retinal. The importance of this visual (retinoid) cycle is underscored by the fact that mutations in genes encoding visual cycle components induce a wide spectrum of diseases characterized by abnormal levels of specific retinoid cycle intermediates. In addition, intense illumination can produce retinoid cycle by-products that are toxic to the retina. Thus, inhibition of the retinoid cycle has therapeutic potential in physiological and pathological states. Four classes of inhibitors that include retinoid and nonretinoid compounds have been identified. We investigated the modes of action of these inhibitors by using purified visual cycle components and in vivo systems. We report that retinylamine was the most potent and specific inhibitor of the retinoid cycle among the tested compounds and that it targets the retinoid isomerase, RPE65. Hydrophobic primary amines like farnesylamine also showed inhibitory potency but a short duration of action, probably due to rapid metabolism. These compounds also are reactive nucleophiles with potentially high cellular toxicity. We also evaluated the role of a specific protein-mediated mechanism on retinoid cycle inhibitor uptake by the eye. Our results show that retinylamine is transported to and taken up by the eye by retinol-binding protein-independent and retinoic acid-responsive gene product 6-independent mechanisms. Finally, we provide evidence for a crucial role of lecithin: retinol acyltransferase activity in mediating tissue specific absorption and long lasting therapeutic effects of retinoid-based visual cycle inhibitors.

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.

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.

Retinoid metabolism (LRAT, REH) in the yolk-sac membrane of Japanese quail eggs and effects of mono- ortho-PCBs

Retinoids stored in the avian egg are essential for normal development, however, laboratory and field experiments suggest that they are affected by environmental contaminants. Lecithin:retinol acyltransferase (LRAT) activity was detected in the microsomal fraction of the yolk-sac membrane of the Japanese quail at day 6 of development. LRAT activity was maximal at pH 7.0 having apparent kinetic parameters of K m=1.35 μM and V max=0.21 nmol/mg protein/h and was inhibited by the sulfhydryl modifying agent N-ethyl-maleimide. Retinol ester hydrolase (REH) activity in the microsomal fraction of the yolk-sac membrane was stimulated by the bile salt analogue 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane sulfonate and was maximal at pH 9.0 with apparent K m=77 μM and V max=34.3 nmol/mg protein/h. Injection of the PCB congener 2,3,3′,4,4′-pentachlorobiphenyl increased both REH and LRAT activities, whereas 2,3,3′,4-tetrachlorobiphenyl stimulated LRAT. Yolk retinol concentration and the molar ratio retinol:retinyl palmitate were lower in the exposed eggs. Yolk retinol concentration decreased as LRAT increased ( R 2=0.89) suggesting that certain PCB congeners may affect vitamin A mobilization in ovo by increasing LRAT activity in the yolk-sac membrane.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Increases Serum and Kidney Retinoic Acid Levels and Kidney Retinol Esterification in the Rat

Halogenatedorganic environmental contaminants such as dioxins are well-known to affect tissue levels of retinoids. To further investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on retinoid homeostasis, adult male Sprague–Dawley rats were killed 1–112 days after a single oral dose of 10 μg TCDD/kg body wt. Additional groups of rats were killed three days after a single oral dose of 0.1, 1, 10, or 100 μg TCDD/kg body wt. Serum and renal retinoic acid levels were measured, as were levels of serum retinol-binding protein (RBP) in liver, kidneys, and serum. Hepatic and renal formation as well as hepatic hydrolysis of retinyl esters were determined, together with hepatic and renal retinoid levels. In addition, one of the retinyl ester hydrolase (REH) activities was investigated in isolated hepatocytes and hepatic stellate cells from rats killed 7 days after a single oral dose of 10 μg TCDD/kg body wt. No increased hepatic REH activity that could explain the decreased hepatic retinyl ester levels following TCDD treatment was found. In the liver, TCDD increased protein levels, but not mRNA levels, of RBP. A causal relationship is suggested for the increased renal lecithin:retinol acyltransferase (LRAT) activity and increased renal retinyl ester levels in TCDD-treated rats. Importantly, TCDD was shown to substantially increase serum and renal levels of retinoic acid. The ability of TCDD to cause increased tissue retinoic acid levels suggests that TCDD may alter the transcription of retinoic acid-responsive genes.

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

The conversion of β-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 β-carotene as a provitamin A. These processes are participated by β-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 β-carotene cleavage enzyme, retinal reductase and retinol esterifying enzymes along the villus-crypt axis in 8-day-old chick duodenum. The β-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 β-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 β-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, typell (CRBPII) as the donor of the substrate.

Esterification and hydrolysis of vitamin A in the liver of brook trout ( Salvelinus fontinalis) and the influence of a coplanar polychlorinated biphenyl

Recent reports of extremely low retinoid stores in fish living in contaminated river systems prompted an initial investigation of the mechanisms of hepatic storage and mobilization in brook trout. Enzyme characterization in microsomes revealed a lecithin:retinol acyltransferase activity (LRAT) optimum in the alkaline range (pH 9.0; V max=0.6 nmol per mg prot. h −1; K m=10.2 μM) which is not known to occur in mammals, in addition to a secondary optimum at pH 6.5 typical of mammals. Acyl CoA:retinol acyltransferase (ARAT) kinetic parameters were quite different to those of mammals. The substrate affinity of trout ARAT ( K m=1.6 μM) was approximately 22-fold greater than that of the rat while maximal velocity ( V max=0.2 nmol per mg prot. h −1) was 18-fold less. Retinyl ester hydrolase activity (REH) was optimal under acid conditions (pH 4.2; V max=6.6 nmol per mg prot. h −1; K m=0.6 mM), was inhibited by a bile salt analogue and was greater in males than females. This REH was tentatively categorized as a bile salt-independent, acid retinyl ester hydrolase (BSI-AREH). REH was inhibited in a dose-dependent manner following in vivo exposure to a representative environmental contaminant the coplanar polychlorinated biphenyl (PCB), 3,3′,4,4′-tetrachlorobiphenyl (TCBP). Inhibition may be an indirect effect because enzyme activity was not affected by in vitro exposure of control microsomes. REH inhibition in the brook trout may affect the uptake of retinyl esters (REs) from chylomicron remnants as well as the mobilization of stored REs.

Reduced lecithin:retinol acyl transferase activity in cultured squamous cell carcinoma lines results in increased substrate-driven retinoic acid synthesis

The uptake and metabolism of retinol was compared in squamous cell carcinoma lines, SCC12b and SCC13, and in normal human keratinocytes (NHK). Long chain fatty acid esters of retinol and 3,4-didehydroretinol were the predominant metabolites formed in both cell types. Lesser amounts of unesterified retinol, 3,4-didehydroretinol, and their respective active acid forms were also observed. Despite a qualitatively similar retinoid composition, there were significant quantitative differences between cell types. Most notable was that SCC formed only about one-fourth the retinoid ester as did normal cells. In parallel with this, unesterified retinol and retinoic acid concentrations in SCC were significantly elevated over those in normal cells. This altered pattern of retinoid metabolites in SCC was found to be due to very low lecithin:retinol acyltransferase (LRAT) activity. SCC exhibited less than one-tenth the LRAT activity of normal cells. Acyl-coenzyme A:retinol acyltransferase (ARAT) and retinyl ester hydrolase activities were not different between cell types. Challenging cells with increasing medium retinol concentrations resulted in dose-dependent increases in retinol and retinoic acid within SCC. In contrast, retinol and retinoic acid concentrations in similarly challenged normal cells remained relatively low across a wide retinol concentration range. This was accomplished by the storage of retinol, via LRAT activity, as retinyl ester. Consistent with increased substrate-driven retinoic acid synthesis in SCC, the expression of transglutaminase 1 was suppressed to a greater extent in the SCCs than in NHK, when cells were exposed to equivalent medium concentrations of retinol. The data demonstrate a central role of LRAT in regulating retinoic acid synthesis via its capacity to modulate cellular levels of substrate retinol.

Retinol esterification activity contributes to retinol transport in stellate cells.

The mechanisms of retinol transport and accumulation in hepatic stellate cells (HSC) remain to be elucidated. Our previous studies suggested that retinol esterification activity, particularly lecithin:retinol acyltransferase (LRAT) activity, in liver retinoid metabolism is important to elucidate the relationship between retinol uptake by HSC and the esterification of retinol. In the present study, using a human HSC-like cell line, LI90, we demonstrated that retinol esterification activity of LI90 cells is similar to that of primary cultures of rat HSC and higher than that of a human hepatoma cell line. Further, since progesterone or diphospho-lauroyl-phosphatidylcholine increased retinol esterification activity of LI90 cells, it is likely that LRAT contributes to retinol esterification in LI90. We examined retinol esterification in LI90 cells and clearance of retinol from culture medium. The percentages of both retinol and esterified retinol in LI90 cells increased in a manner dependent on retinol concentration in medium, whereas that of retinol in medium decreased. The percentages of esterified and unesterified retinol in LI90 cells and of retinol in medium were linearly dependent on the logarithm of the initial concentration of retinol in the medium. These results suggest that retinol esterification activity contributes to retinol uptake by HSC and maintenance of non-toxic retinol levels in plasma.

Regulation of Hepatic Lecithin:Retinol Acyltransferase Activity by Retinoic Acid Receptor-Selective Retinoids

The microsomal enzyme LRAT esterifies retinol and has been implicated in the hepatic storage of vitamin A. Previously, we showed that hepatic LRAT activity is negligible during vitamin A deficiency and that all-trans-retinoic acid (all-trans-RA) rapidly induces the activity of liver LRAT in retinoid-deficient rats. In the present studies, we have examined the ability of natural and synthetic retinoids to induce liver LRAT activity in retinoid-deficient rats. The natural retinoids retinol, all-trans-RA (100 μg), 9-cis-RA, or equal molar amounts of other retinoids were injected ip and LRAT specific activity was measured in liver homogenates 17–18 h later. In retinoid-deficient rats, liver LRAT activity was extremely low [0.13 ± 0.03 pmol retinyl ester (RE)/min/mg liver protein, mean ± SE]. The natural retinoids retinol and all-trans-RA strongly induced LRAT activity (12.71 ± 1.09 and 13.10 ± 1.55 pmol RE/min/mg, respectively), whereas 9-cis-RA induced a lower level of LRAT activity (3.96 ± 1.88 pmol RE/min/mg,P< 0.001 vs all-trans-RA). The retinoic acid receptor (RAR)-selective analog (RAR pan-agonist) all-trans-UAB8 and the RAR-α-selective retinoid Am580 also strongly induced LRAT activity. In contrast, neither RXR-selective agonists nor retinoids having aretrostructure were active. For retinoids with significant RAR-α binding activity there was a strong direct correlation between receptor bindingin vitroand the ability to induce hepatic LRAT activityin vivo(r2= 0.920). These data implicate the RARs in the induction of hepatic LRAT and suggest a predominant role for RAR-α-active ligands.

Hepatic lecithin: Retinol acyltransferase activity is induced in vivo by retinoic acid, but not by triiodothyronine, in vitamin A-deficient, hypothyroid rats

The activity of the enzyme lecithin; retinol acyltransferase (LRAT) is extremely low in the liver of vitamin A-deficient rats, but is rapidly induced after administration of retinoic acid (RA). The nuclear receptors for RA are closely related to the receptors for thyroid hormone, and molecular cross-talk between these receptors has been observed in vitro and in cultured cells. Therefore we have examined whether retinoid status and thyroid hormone status interact in the regulation of hepatic LRAT activity in vivo. Vitamin A-deficient male Lewis rats, either euthyroid or made hypothyroid by treatment with propylthiouracil, were treated once or three times with 20 μg of all- trans-RA, 10 μg/100 gm body weight of triiodothyronine (T 3), or both hormones. Hepatic LRAT activity, which was negligible in all retinoid-deficient rats, was induced by RA ( P < 0.0001) regardless of the animal's thyroid hormone status. T 3 by itself had no ability to induce hepatic LRAT activity ( P = 0.42), nor did co-administration of T 3 with RA increase or decrease the response to RA ( P = 0.13). In retinoid-sufficient rats, hypothyroidism did not alter hepatic LRAT activity; however, LRAT activity was reduced by half ( P < 0.05) after three treatments with T 3. Therefore we conclude that thyroid hormone status alone does not regulate LRAT, nor does thyroid status affect the ability of RA to induce hepatic LRAT in retinoid-deficient animals. However, in retinoid-sufficiency, repeated treatment with T 3 may reduce the hepatic esterification of retinol by LRAT.

Lecithin:Retinol Acyltransferase and Retinyl Ester Hydrolase Activities Are Differentially Regulated by Retinoids and Have Distinct Distributions between Hepatocyte and Nonparenchymal Cell Fractions of Rat Liver ,

The cellular distribution of enzymes that esterify retinol and hydrolyze retinyl esters (RE) was studied in liver of vitamin A–sufficient,–deficient, and deficient rats treated with retinoic acid or N-(4-hydroxyphenyl)-retinamide. Livers were perfused and cell fractions enriched in hepatocytes, and nonparenchymal cells were obtained for assays of RE and enzyme activity. The specific activity of lecithin:retinol acyltransferase (LRAT) was approximately 10-fold greater in the nonparenchymal cell than the hepatocyte fraction from both vitamin A–sufficient and retinoid-treated rats. Total RE mass, newly synthesized [3H]RE and LRAT activity were positively correlated in liver and isolated cells of both normal (P < 0.0001) and retinoid-treated rats (P < 0.0002). In nonparenchymal cells, these three constituents were nearly equally enriched as evaluated by their relative specific activity values (RSA, defined as the percentage of recovered activity divided by the percentage of recovered protein), which were each significantly greater than 1.0, with values of 4.3 for total RE mass (P < 0.05), 3.6 for newly synthesized [3H]RE (P < 0.01) and 3.8 for LRAT activity (P < 0.01). In contrast, the specific activities of neutral and acid bile salt–independent retinyl ester hydrolases (REH) did not vary with vitamin A status, and their RSA values were close to 1.0 in both hepatocytes and nonparenchymal cells. These data show that LRAT and REH are differentially regulated by retinoids and that these enzymes also differ in their spacial distribution between liver parenchymal and nonparenchymal cells.

N-(4-Hydroxyphenyl)-Retinamide Increases Lecithin:Retinol Acyltransferase Activity in Rat Liver

N-(4-Hydroxyphenyl)-retinamide (4-HPR; Fenretinide) is a synthetic retinoid which is undergoing investigation as a cancer chemopreventive agent. However, 4-HPR alters vitamin A kinetics and reduces the concentration of plasma retinol. We have conducted studies to examine the effects of 4-HPR on the activity of the enzyme lecithin:retinol acyltransferase (LRAT). This enzyme is implicated in the absorption and storage of vitamin A and is regulated, in liver, by vitamin A nutritional status. To determine whether 4-HPR, like retinoic acid, is able to induce liver LRAT activity, vitamin A-deficient rats having negligible liver LRAT activity were treated with single doses of 4-HPR (0.02–2.5 mg) and liver homogenates were assayed for LRAT activity using 3H-retinol bound to the cellular-retinol binding protein, CRBP, as substrate. Treatment with 4-HPR resulted in a dose- and time-dependent increase in liver LRAT activity which reached a maximum at 24 h. The activity of LRAT assayed in vitro and of hepatic 13 H-retinyl ester content determined after an in vivo pulse of 3H-retinol were highly correlated (r = 0.802, P < 0.0002). When vitamin A-sufficient rats were fed a 4-HPR-supplemented diet for 30 d, LRAT activity differed significantly from control values in the liver (P < 0.0001) but not the small intestines. Changes in hepatic retinol metabolism which favor the esterification of vitamin A may be related to the mechanism by which 4-HPR alters vitamin A kinetics in vivo.

Lack of lecithin: retinol acyltransferase activity in chick lungs.

Our previous study revealed that no retinyl esters were detectable in chick and hen lungs, suggesting that the retinol esterification system may be absent in these tissues. This possibility encouraged us to investigate whether chick lungs exhibit the activity of a retinol esterifying enzyme, i.e., lecithin: retinol acyltransferase (LRAT). The LRAT activity was assayed with dilauroyl phosphatidylcholine and either complex of retinol-cellular retinol-binding protein, type two or retinol-cellular retinol-binding protein in microsomal preparations of lung, duodenum and liver of 7-day-old chicks. Relatively high levels of LRAT activity were present in the duodenum and the liver of chicks as well as in the rat lung. However, the chick lung exhibited no LRAT activity. The lungs of both rat and chick showed similar and low levels of acyl-CoA: retinol acyltransferase (ARAT) activity, but only rat lung, but not chick lung, contained a detectable amount of retinyl esters. Thus, the retinyl ester storage in the lung seems to depend on the presence of LRAT activity in the lung, but it is independent of the presence of ARAT activity in the lung. The absence of LRAT activity and retinyl esters in the chick lung suggests that the retinol in the chick lung may not be provided from retinyl ester storage, and the retinol transferred directly from serum should be utilized to generate retinoic acid.

Amount and quality of dietary protein regulate lecithin:retinol acyltransferase activity without change in cellular retinol-binding protein, type two in rat jejunum

The effects of amount and quality of dietary protein on the cellular retinol-binding protein (type two) and two enzymes for retinol esterification, i.e., lecithin:retinol acyltransferase and acyl-CoA:retinol acyltransferase in rat jejunum were examined. Three groups of five rats were pair-fed defined diets for 14 days. The control group was fed a diet containing 20% casein; the two experimental groups received diets containing 5% casein or 20% gluten. Feeding the 5% casein diet or the 20% gluten diet decreased serum concentration of retinol and retinol-binding protein and increased liver total retinol and retinol-binding protein contents. The lecithin:retinol acyltransferase activity in jejunal microsomes, which was determined using retinol bound to cellular retinol-binding protein (type two), was significantly reduced in animals fed the 20% gluten diet (by 30%) as compared with animals fed the 20% casein diet. Feeding the 5% casein diet also led to a slight decrease in the segmental lecithin:retinol acyltransferase activity. The acyl-CoA:retinol acyltransferase activity was unaffected by feeding these diets. The jejunal cytosolic cellular retinol-binding protein (type two) content was also unchanged by feeding these diets. Feeding the 20% wheat gluten diet resulted in remarkable low levels of the unesterified retinol (by 64%), retinyl palmitate (by 32%), and protein (by 47%) in the whole jejunal mucosa, when compared with the corresponding values of the 20% casein group. These results suggest that the dietary protein malnutrition will suppress intestinal esterification of retinol absorbed by a decline of lecithin:retinol acyltransferase activity and without change in intestinal cellular retinol-binding protein (type two) level. The results also suggest that retinol uptake may decrease due to an impaired absorption process in protein malnutritional status.

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.

Regulation of Hepatic Lecithin: Retinol Acyltransferase Activity by Retinoic Acid

The activity of the hepatic enzyme lecithin: retinol acyltransferase (LRAT), thought to catalyze the esterification of retinol for storage, was previously shown to vary directly with the vitamin A (retinol) status of the rat [Randolph, R. K., and Ross, A. C. (1991) J. Biol. Chem. 266, 16453-16457]. The present studies were designed to determine whether liver LRAT activity is regulated in vivo by retinoic acid, a principal active metabolite of retinol. LRAT activity was negligible in the livers of vitamin A-deficient rats. Following treatment with a single 2-μg dose of retinoic acid, LRAT activity increased significantly while, following treatment with a single 20-μg dose, liver LRAT activity equalled that of vitamin A-sufficient adult rats. Retinoic acid was more effective than an equimolar quantity of retinol in restoring LRAT activity in the vitamin A-deficient rat liver. The increase in hepatic LRAT activity after administration of retinoic acid occurred rapidly, reaching a maximum within 12-16 h but declining again after 48-72 h. Studies were conducted in vivo to gain insight into the level of regulation of LRAT by retinoic acid. The increase in LRAT activity by retinoic acid in the vitamin A-deficient rat was blocked completely by both actinomycin D and cycloheximide. The ability of liver to esterify retinol in vivo was correlated with the in vitro activity of LRAT after retinoic acid induction. We conclude that retinoic acid, an important end product of retinol metabolism, regulates a key aspect of hepatic retinal metabolism through its regulatory activity on liver LRAT.