Retinyl Ester Hydrolase Research Articles

Lysosomal acid lipase is the major acid retinyl ester hydrolase in cultured human hepatic stellate cells but not essential for retinyl ester degradation

Vitamin A is stored as retinyl esters (REs) in lipid droplets of hepatic stellate cells (HSCs). To date, two different pathways are known to facilitate the breakdown of REs: (i) Hydrolysis of REs by neutral lipases, and (ii) whole lipid droplet degradation in autolysosomes by acid hydrolysis.In this study, we evaluated the contribution of neutral and acid RE hydrolases to the breakdown of REs in human HSCs. (R)-Bromoenol lactone (R-BEL), inhibitor of adipose triglyceride lipase (ATGL) and patatin-like phospholipase domain-containing 3 (PNPLA3), the hormone-sensitive lipase (HSL) inhibitor 76-0079, as well as the serine-hydrolase inhibitor Orlistat reduced neutral RE hydrolase activity of LX-2 cell-lysates between 20 and 50%. Interestingly, in pulse-chase experiments, R-BEL, 76-0079, as well as Orlistat exerted little to no effect on cellular RE breakdown of LX-2 cells as well as primary human HSCs. In contrast, Lalistat2, a specific lysosomal acid lipase (LAL) inhibitor, virtually blunted acid in vitro RE hydrolase activity of LX-2 cells. Accordingly, HSCs isolated from LAL-deficient mice showed RE accumulation and were virtually devoid of acidic RE hydrolase activity. In pulse-chase experiments however, LAL-deficient HSCs, similar to LX-2 cells and primary human HSCs, were not defective in degrading REs.In summary, results demonstrate that ATGL, PNPLA3, and HSL contribute to neutral RE hydrolysis of human HSCs. LAL is the major acid RE hydrolase in HSCs. Yet, LAL is not limiting for RE degradation under serum-starvation. Together, results suggest that RE breakdown of HSCs is facilitated by (a) so far unknown, non-Orlistat inhibitable RE-hydrolase(s).

Pancreatic lipase-related protein 2 is responsible for the increased hepatic retinyl ester hydrolase activity in vitamin A-deficient mice.

The formation and hydrolysis of hepatic retinyl esters (RE) is a key process inmaintaining serum retinol homeostasis. During vitamin A deficiency, the activity of RE hydrolases (REH) in liver increases to cope with the reduced dietary vitamin A intake. However, it remains unclear which REH is the main enzyme responsible for RE hydrolysis in the liver under physiological conditions. Our previous studies have shown that pancreatic lipase-related protein 2 (PLRP2) is conditionally expressed in the liver and may be involved in the hydrolysis of hepatic RE. In the current study, we generated Plrp2-/- mice using transcription activator-like effector nuclease technology to investigate the role of PLRP2 in the metabolism of hepatic RE. Compared with the mice fed normal diet, the hepatic REH activity of wild-type (WT) mice fed vitamin A-deficient diet (VAD) increased significantly, while this activity did not increase in Plrp2-/- mice fed VAD. Plrp2-/- mice showed higher residual RE content in liver and lower serum retinol level, compared with WT mice fed VAD. Hepatic metabolic profiling from 1 H NMR-based metabolomics suggested that Plrp2-/- mice were more sensitive to VAD. Docking analysis and enzyme activity assay revealed that retinyl palmitate was the substrate with higher affinity for PLRP2. Our results indicate that Plrp2 can be activated in the liver and is responsible for the increased REH activity in the liver of mice fed VAD.

Hormone-sensitive lipase is a retinyl ester hydrolase in human and rat quiescent hepatic stellate cells

Hepatic stellate cells (HSC) store vitamin A as retinyl esters and control circulating retinol levels. Upon liver injury, quiescent (q)HSC lose their vitamin A and transdifferentiate to myofibroblasts, e.g. activated (a)HSC, which promote fibrosis by producing excessive extracellular matrix. Adipose triglyceride lipase/patatin-like phospholipase domain-containing protein 2 (ATGL/PNPLA2) and adiponutrin (ADPN/PNPLA3) have so far been shown to mobilize retinol from retinyl esters in HSC. Here, we studied the putative role of hormone-sensitive lipase (HSL/LIPE) in HSC, as it is the major retinyl ester hydrolase (REH) in adipose tissue.Lipe/HSL expression was analyzed in rat liver and primary human and rat qHSC and culture-activated aHSC. Retinyl hydrolysis was analyzed after Isoproterenol-mediated phosphorylation/activation of HSL.Primary human HSC contain 2.5-fold higher LIPE mRNA levels compared to hepatocytes. Healthy rat liver contains significant mRNA and protein levels of HSL/Lipe, which predominates in qHSC and cells of the portal tree. Q-PCR comparison indicates that Lipe mRNA levels in qHSC are dominant over Pnpla2 and Pnpla3. HSL is mostly phosphorylated/activated in qHSC and partly colocalizes with vitamin A-containing lipid droplets. Lipe/HSL and Pnpla3 expression is rapidly lost during HSC culture-activation, while Pnpla2 expression is maintained. HSL super-activation by isoproterenol accelerates loss of lipid droplets and retinyl palmitate from HSC, which coincided with a small, but significant reduction in HSC proliferation and suppression of Collagen1A1 mRNA and protein levels.In conclusion, HSL participates in vitamin A metabolism in qHSC. Equivalent activities of ATGL and ADPN provide the healthy liver with multiple routes to control circulating retinol levels.

Disturbed Vitamin A Metabolism in Non-Alcoholic Fatty Liver Disease (NAFLD).

Vitamin A is required for important physiological processes, including embryogenesis, vision, cell proliferation and differentiation, immune regulation, and glucose and lipid metabolism. Many of vitamin A’s functions are executed through retinoic acids that activate transcriptional networks controlled by retinoic acid receptors (RARs) and retinoid X receptors (RXRs).The liver plays a central role in vitamin A metabolism: (1) it produces bile supporting efficient intestinal absorption of fat-soluble nutrients like vitamin A; (2) it produces retinol binding protein 4 (RBP4) that distributes vitamin A, as retinol, to peripheral tissues; and (3) it harbors the largest body supply of vitamin A, mostly as retinyl esters, in hepatic stellate cells (HSCs). In times of inadequate dietary intake, the liver maintains stable circulating retinol levels of approximately 2 μmol/L, sufficient to provide the body with this vitamin for months. Liver diseases, in particular those leading to fibrosis and cirrhosis, are associated with impaired vitamin A homeostasis and may lead to vitamin A deficiency. Liver injury triggers HSCs to transdifferentiate to myofibroblasts that produce excessive amounts of extracellular matrix, leading to fibrosis. HSCs lose the retinyl ester stores in this process, ultimately leading to vitamin A deficiency. Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and is a spectrum of conditions ranging from benign hepatic steatosis to non-alcoholic steatohepatitis (NASH); it may progress to cirrhosis and liver cancer. NASH is projected to be the main cause of liver failure in the near future. Retinoic acids are key regulators of glucose and lipid metabolism in the liver and adipose tissue, but it is unknown whether impaired vitamin A homeostasis contributes to or suppresses the development of NAFLD. A genetic variant of patatin-like phospholipase domain-containing 3 (PNPLA3-I148M) is the most prominent heritable factor associated with NAFLD. Interestingly, PNPLA3 harbors retinyl ester hydrolase activity and PNPLA3-I148M is associated with low serum retinol level, but enhanced retinyl esters in the liver of NAFLD patients. Low circulating retinol in NAFLD may therefore not reflect true “vitamin A deficiency”, but rather disturbed vitamin A metabolism. Here, we summarize current knowledge about vitamin A metabolism in NAFLD and its putative role in the progression of liver disease, as well as the therapeutic potential of vitamin A metabolites.

Hepatic Retinyl Ester Hydrolases and the Mobilization of Retinyl Ester Stores.

For mammals, vitamin A (retinol and metabolites) is an essential micronutrient that is required for the maintenance of life. Mammals cannot synthesize vitamin A but have to obtain it from their diet. Resorbed dietary vitamin A is stored in large quantities in the form of retinyl esters (REs) in cytosolic lipid droplets of cells to ensure a constant supply of the body. The largest quantities of REs are stored in the liver, comprising around 80% of the body’s total vitamin A content. These hepatic vitamin A stores are known to be mobilized under times of insufficient dietary vitamin A intake but also under pathological conditions such as chronic alcohol consumption and different forms of liver diseases. The mobilization of REs requires the activity of RE hydrolases. It is astounding that despite their physiological significance little is known about their identities as well as about factors or stimuli which lead to their activation and consequently to the mobilization of hepatic RE stores. In this review, we focus on the recent advances for the understanding of hepatic RE hydrolases and discuss pathological conditions which lead to the mobilization of hepatic RE stores.

Mechanical force and tensile strain activated hepatic stellate cells and inhibited retinol metabolism.

Hepatic stellate cells (HSCs), the principal producers of extracellular matrix proteins, play a major role in the development of liver fibrosis which is accompanied with elevated sinusoidal pressure and portal hypertension. We have isolated primary rat HSCs and investigated the effect of mechanical pressure and tensile strain on retinol metabolism in the cells. Mechanical force and tensile strain significantly increased the expression of α-smooth muscle actin (α-SMA) and collagen I, and notably inhibited the expression of cellular retinol-binding protein I (CRBP-I), lecithin-retinol acyltransferase (LRAT), retinyl ester hydrolase (REH), retinoic acid receptor-β (RAR-β) and retinoid X receptor-α (RXR-α). Such effects were partially reversed by the retinoid X receptor antagonist, HX531, and the retinoic acid receptor antagonist, LE135. Mechanical force and tensile strain significantly activate HSCs by regulating the retinoid metabolic pathway. Activation of HSCs can therefore be manipulated through mechanical force and tensile strain in vitro.

Adipose triglyceride lipase is involved in the mobilization of triglyceride and retinoid stores of hepatic stellate cells

Hepatic stellate cells (HSCs) store triglycerides (TGs) and retinyl ester (RE) in cytosolic lipid droplets. RE stores are degraded following retinoid starvation or in response to pathogenic stimuli resulting in HSC activation. At present, the major enzymes catalyzing lipid degradation in HSCs are unknown. In this study, we investigated whether adipose triglyceride lipase (ATGL) is involved in RE catabolism of HSCs. Additionally, we compared the effects of ATGL deficiency and hormone-sensitive lipase (HSL) deficiency, a known RE hydrolase (REH), on RE stores in liver and adipose tissue. We show that ATGL degrades RE even in the presence of TGs, implicating that these substrates compete for ATGL binding. REH activity was stimulated and inhibited by comparative gene identification-58 and G0/G1 switch gene-2, respectively, the physiological regulators of ATGL activity. In cultured primary murine HSCs, pharmacological inhibition of ATGL, but not HSL, increased RE accumulation. In mice globally lacking ATGL or HSL, RE contents in white adipose tissue were decreased or increased, respectively, while plasma retinol and liver RE levels remained unchanged. In conclusion, our study shows that ATGL acts as REH in HSCs promoting the degradation of RE stores in addition to its established function as TG lipase. HSL is the predominant REH in adipocytes but does not affect lipid mobilization in HSCs.

Waterborne cadmium and nickel impact oxidative stress responses and retinoid metabolism in yellow perch

In this experiment, we studied the transcriptional and functional (enzymatic) responses of yellow perch (Perca flavescens) to metal stress, with a focus on oxidative stress and vitamin A metabolism. Juvenile yellow perch were exposed to two environmentally relevant concentrations of waterborne cadmium (Cd) and nickel (Ni) for a period of 6 weeks. Kidney Cd and Ni bioaccumulation significantly increased with increasing metal exposure. The major retinoid metabolites analyzed in liver and muscle decreased with metal exposure except at high Cd exposure where no variation was reported in liver. A decrease in free plasma dehydroretinol was also observed with metal exposure. In the liver of Cd-exposed fish, both epidermal retinol dehydrogenase 2 transcription level and corresponding enzyme activities retinyl ester hydrolase and lecithin dehydroretinyl acyl transferase increased. In contrast, muscle epidermal retinol dehydrogenase 2 transcription level decreased with Cd exposure. Among antioxidant defences, liver transcription levels of catalase, microsomal glutathione-S-transferase-3 and glucose-6-phosphate dehydrogenase were generally enhanced in Cd-exposed fish and this up-regulation was accompanied by an increase in the activities of corresponding enzymes, except for microsomal glutathione-S-transferase. No consistent pattern in antioxidant defence responses was observed between molecular and biochemical response when fish were exposed to Ni, suggesting a non-synchronous response of antioxidant defence in fish exposed to waterborne Ni. There was a general lack of consistency between muscle transcription level and enzyme activities analyzed. The overall findings from this investigation highlight the usefulness of transcriptional and biochemical endpoints in the identification of oxidative stress and vitamin A metabolism impairment biomarkers and the potential use of multi-level biological approaches when assessing environmental risk in fish.

Vitamin A and vitamin E levels in lung from subjects with chronic obstructive pulmonary disease with different degree of severity of emphysema

Vitamin A (VA) and its active metabolites play an essential role in the respiratory tract by influencing differentiation and the integrity of the epithelium. A study that attempted to relate VA status with the degree of lung airway obstruction, revealed a lower serum retinol concentration in patients with moderate to severe COPD. Further, treatment with retinol improved the FEV1 in subjects with COPD.To test our hypothesis that VA signaling pathway is compromised in COPD, we analyzed human lung samples from COPD patients, differing in the degree of severity of emphysema, provided by the Lung Tissue Research Consortium for their VA and tocopherol (T)‐levels by HPLC. T levels were determined since it is known that α–T is an inhibitor of retinyl ester hydrolase activity. VA levels (retinol and retinyl ester) were found to decrease with severity of the disease from mild (168 ng/g, 160 ng/g), to moderate (136 ng/g, 99 ng/g), and to severe (127 ng/g, 71 ng/g). α‐T levels were less in moderate (12.8 μg/g) and severe (13.7 μg/g) groups than that in mild group (15.5 μg/g). Similarly δ‐T‐levels were less in moderate (229 ng/g) and severe (142 ng/g) groups than that in mild group (231 ng/g). However, it is interesting to note that γ‐T levels were higher in moderate (4.6 μg/g) and severe (2.2 μg/g) groups than that in mild group (1.3 μg/g). Results indicate an involvement of VA & Ts in COPD. (Supported by NIH grant 1RO3 HL095419‐01A1).

Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver

Excess dietary vitamin A is esterified with fatty acids and stored in the form of retinyl ester (RE) predominantly in the liver. According to the requirements of the body, liver RE stores are hydrolyzed and retinol is delivered to peripheral tissues. The controlled mobilization of retinol ensures a constant supply of the body with the vitamin. Currently, the enzymes catalyzing liver RE hydrolysis are unknown. In this study, we identified mouse esterase 22 (Es22) as potent RE hydrolase highly expressed in the liver, particularly in hepatocytes. The enzyme is located exclusively at the endoplasmic reticulum (ER), implying that it is not involved in the mobilization of RE present in cytosolic lipid droplets. Nevertheless, cell culture experiments revealed that overexpression of Es22 attenuated the formation of cellular RE stores, presumably by counteracting retinol esterification at the ER. Es22 was previously shown to form a complex with beta-glucuronidase (Gus). Our studies revealed that Gus colocalizes with Es22 at the ER but does not affect its RE hydrolase activity. Interestingly, however, Gus was capable of hydrolyzing the naturally occurring vitamin A metabolite retinoyl beta-glucuronide. In conclusion, our observations implicate that both Es22 and Gus play a role in liver retinoid metabolism.

Cellular retinol-binding protein functions in the regulation of retinoid metabolism.

A microsomal retinyl ester hydrolase from rat tissues is activated by apo-cellular retinol-binding protein, which thus regulates the availability of retinol either for binding to serum retinol-binding protein or for metabolic oxidation. Microsomal retinal synthesis was found to occur in rat tissues with holo-cellular retinol-binding protein as substrate. Retinal could be further oxidized to retinoic acid by a cytosolic fraction.

Hormone‐sensitive lipase (HSL) is also a retinyl ester hydrolase: evidence from mice lacking HSL

Here, we investigated the importance of hormone-sensitive lipase (HSL) as a retinyl ester hydrolase (REH). REH activity was measured in vitro using recombinant HSL and retinyl palmitate. The expression of retinoic acid (RA)-regulated genes and retinoid metabolites were measured in high-fat diet fed HSL-null mice using real-time quantitative PCR and triple-stage liquid chromatography/tandem mass spectrometry, respectively. Age- and gender-matched wild-type littermates were used as controls. The REH activity of rat HSL was found to be higher than that against the hitherto best known HSL substrate, i.e., diacylglycerols. REH activity in white adipose tissue (WAT) of HSL-null mice was completely blunted and accompanied by increased levels of retinyl esters and decreased levels of retinol, retinaldehyde and all-trans RA. Accordingly, genes known to be positively regulated by RA were down-regulated in HSL-null mice, including pRb and RIP140, key factors promoting differentiation into the white over the brown adipocyte lineage. Dietary RA supplementation partly restored WAT mass and the expression of RA-regulated genes in WAT of HSL-null mice. These findings demonstrate the importance of HSL as an REH of adipose tissue and suggest that HSL via this action provides RA and other retinoids for signaling events that are crucial for adipocyte differentiation and lineage commitment.

Expression of carboxylesterase and lipase genes in rat liver cell-types

Approximately 80% of the body vitamin A is stored in liver stellate cells with in the lipid droplets as retinyl esters. In low vitamin A status or after liver injury, stellate cells are depleted of the stored retinyl esters by their hydrolysis to retinol. However, the identity of retinyl ester hydrolase(s) expressed in stellate cells is unknown. The expression of carboxylesterase and lipase genes in purified liver cell-types was investigated by real-time PCR. We found that six carboxylesterase and hepatic lipase genes were expressed in hepatocytes. Adipose triglyceride lipase was expressed in Kupffer cells, stellate cells and endothelial cells. Lipoprotein lipase expression was detected in Kupffer cells and stellate cells. As a function of stellate cell activation, expression of adipose triglyceride lipase decreased by twofold and lipoprotein lipase increased by 32-fold suggesting that it may play a role in retinol ester hydrolysis during stellate cell activation.

Influence of iron on vitamin A nutritional status

Iron deficiency seems to deteriorate vitamin A metabolism leading to a reduction in serum retinol and an increase in hepatic retinol and retinyl ester. These alterations probably result from an increase in retinol sequestration to the liver and/or impairment in the activity of hepatic retinyl ester hydrolases decreasing vitamin A mobilization.

Molecular Screening for GS2 Lipase Regulators: Inhibition of Keratinocyte Retinylester Hydrolysis by TIP47

Retinoic acid at nanomolar concentrations modulates epidermal functions by serving as a transcription factor ligand. Under conditions of retinol sufficiency, it is imperative to limit retinoic acid biosynthesis from serum-derived retinol. In the epidermis, this is accomplished by esterifying retinol with long-chain fatty acids. Retinylester (RE) pools serve as a source of retinol for retinoic acid production under retinol deficiency and when required for proper differentiation. We have recently reported that GS2 lipase is expressed in keratinocytes and has the enzymatic properties of keratinocyte RE hydrolase. As GS2 lipase has a robust activity that can affect the intracellular retinol levels, we postulated that its activity must be regulated. Therefore, we screened keratinocyte cDNA expression libraries for the putative inhibitor. Herein, we report the identity of an inhibitor, TIP47, which prevents RE hydrolysis catalyzed by GS2 lipase and hormone-sensitive lipase. This protein was known to transport mannose-6-phosphate receptors from endosome to trans-Golgi and to be distributed between the cytoplasm and lipid droplets. Using a series of deletion mutants, we found two regions involved in the inhibitory activity. Residues within the carboxyl alpha3-alpha4 helices are essential in the context of the full-length protein. Residues within the amino-terminal also contribute depending on the context.

A comparative study on the effects of oral amiodarone and trimeprazine, twoin vitroretinyl ester hydrolase inhibitors, on the metabolic availability of vitamin A in rats

Amiodarone, an antiarrhythmic drug, and trimeprazine, an antipsychotic drug, are both in vitro inhibitors of retinyl ester hydrolase. To determine whether these agents have deleterious effects on aspects of vitamin A metabolism, Brown Norway rats (n 18) were treated at clinically equivalent doses once daily for 26 d with either oral drug. On day 27, a tolerance test was used to determine whether these agents interfered with vitamin absorption. During the first 8 d, the plasma retinol level declined in all animals. Between days 12 and 27, it rose to near pre-treatment concentrations in the control and trimeprazine groups and remained relatively constant at low levels (P<0.001) in the amiodarone group. The intestinal absorption of vitamin A was reduced (P<0.05) in the amiodarone group compared with the placebo and trimeprazine groups, which did not differ significantly from each other. At the end of the 4-week treatment period, hepatic retinyl ester hydrolase activity was lower in the drug-dosed rats (P=0.06 for amiodarone) than in the controls. With regard to effects on liver reserves, drug treatment resulted in vitamin A depletion (P<0.019), and distinctive patterns of retinol and its esters were seen in response to dosing. In conclusion, amiodarone and trimeprazine have been shown to influence different aspects of retinoid metabolism, namely absorption, storage and transport. In clinical practice, the routine unmonitored use of these drugs and the suggestion that these agents be taken with meals are not recommended.

Isolation and Characterization of a Microsomal Acid Retinyl Ester Hydrolase

Previous work demonstrated both acid and neutral, bile salt-independent retinyl ester hydrolase activities in rat liver homogenates. Here we present the purification, identification, and characterization of an acid retinyl ester hydrolase activity from solubilized rat liver microsomes. Purification to homogeneity was achieved by sequential chromatography using SP-Sepharose cation exchange, phenyl-Sepharose hydrophobic interaction, concanavalin A-Sepharose affinity and Superose 12 gel filtration chromatography. The isolated protein had a monomer molecular mass of approximately 62 kDa, as measured by mass spectrometry. Gel filtration chromatography of the purified protein revealed a native molecular mass of approximately 176 kDa, indicating that the protein exists as a homotrimeric complex in solution. The purified protein was identified as carboxylesterase ES-10 (EC 3.1.1.1) by N-terminal Edman sequencing and extensive LC-MS/MS sequence analysis and cross-reaction with an anti-ES-10 antibody. Glycosylation analysis revealed that only one of two potential N-linked glycosylation sites is occupied by a high mannose-type carbohydrate structure. Using retinyl palmitate in a micellar assay system the enzyme was active over a broad pH range and displayed Michaelis-Menten kinetics with a K(m) of 86 microm. Substrate specificity studies showed that ES-10 is also able to catalyze hydrolysis of triolein. Cholesteryl oleate was not a substrate for ES-10 under these assay conditions. Real time reverse transcriptase-PCR and Western blot analysis revealed that ES-10 is highly expressed in liver and lung. Lower levels of ES-10 mRNA were also found in kidney, testis, and heart. A comparison of mRNA expression levels in liver demonstrated that ES-10, ES-4, and ES-3 were expressed at significantly higher levels than ES-2, an enzyme previously thought to play a major role in retinyl ester metabolism in liver. Taken together these data indicate that carboxylesterase ES-10 plays a major role in the hydrolysis of newly-endocytosed, chylomicron retinyl esters in both neutral and acidic membrane compartments of liver cells.

Identification of a Novel Keratinocyte Retinyl Ester Hydrolase as a Transacylase and Lipase

Retinoic acid influences epidermal morphology and function through its ability to control transcription. Because the circulation presents the epidermis with micromolar amounts of retinol that can be converted to retinoic acid, regulating retinol access is imperative. In keratinocytes the majority of retinol is sequestered as long chain fatty acid esters. Although much has been learned about the major esterifying enzyme, little is known about the hydrolase that accesses retinol from its storage depot. Murine carboxylesterases and hormone sensitive lipase have been shown to have this activity. We found that their in vitro sensitivity to bis-p-nitrophenyl phosphate (BNPP), however, was not shared by the epidermal hydrolase activity. We therefore produced and screened two keratinocyte cDNA expression libraries and identified a previously sequenced gene (GS2) as a keratinocyte retinyl ester (RE) hydrolase insensitive to BNPP. The enzyme also catalyzes fattyacyl CoA-dependent and -independent retinol esterification. The hydrolysis reaction is greater at neutral pH, whereas the esterification reaction is greater at acidic pH. These activities are consistent with the increased RE content that accompanies epidermal maturation. In addition, this enzyme utilizes triolein as substrate and generates diacylglyceride and free fatty acid.

Flupenthixol and cefotiam: effects on vitamin A metabolism in rats

We examined the alterations in vitamin A metabolism as a result of flupenthixol or cefotiam administration. The impact of these drugs on indices of vitamin A status was evaluated in Brown Norway and Long-Evans rats. Intramuscular drug administration for 28 d resulted in a decline in systemic retinol. Changes in circulating retinol with time for chronic dosing showed drug treatment (P<0.001) and time (P<0.03) to be significant factors, but rat strain (P=0.33) was not a significant factor. Flupenthixol was the most active retinol-lowering compound (P<0.005). At the end of the 28 d period, hepatic retinyl ester hydrolase activity was greater in drug-treated rats than in controls (P<0.05). With regard to effects on liver reserves: (1) flupenthixol treatment resulted in vitamin A depletion (P<0.05); (2) cefotiam treatment stimulated vitamin A accumulation; (3) distinctive patterns of retinol and its esters were seen in response to treatment. It is reasonable to assume that the drugs interfere with vitamin A in at least two ways: (1) lowering of plasma retinol, an early event in the interaction, may be caused by inhibition of hepatic holo-retinol-binding protein secretion or stimulation of clearance, or both; (2) when plasma retinol levels are persistently low, and as the hepatic deposits of the xenobiotics build up, there are changes in the vitamin A pool size and composition of the liver. Candidate enzymes are retinyl ester hydrolase and cytochrome P450. The relationship between these two events will be studied in further detail.

Lecithin-retinol acyltransferase is essential for accumulation of all-trans-retinyl esters in the eye and in the liver.

Lecithin-retinol acyltransferase (LRAT), an enzyme present mainly in the retinal pigmented epithelial cells and liver, converts all-trans-retinol into all-trans-retinyl esters. In the retinal pigmented epithelium, LRAT plays a key role in the retinoid cycle, a two-cell recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. We disrupted mouse Lrat gene expression by targeted recombination and generated a homozygous Lrat knock-out (Lrat-/-) mouse. Despite the expression of LRAT in multiple tissues, the Lrat-/- mouse develops normally. The histological analysis and electron microscopy of the retina for 6-8-week-old Lrat-/- mice revealed that the rod outer segments are approximately 35% shorter than those of Lrat+/+ mice, whereas other neuronal layers appear normal. Lrat-/- mice have trace levels of all-trans-retinyl esters in the liver, lung, eye, and blood, whereas the circulating all-trans-retinol is reduced only slightly. Scotopic and photopic electroretinograms as well as pupillary constriction analyses revealed that rod and cone visual functions are severely attenuated at an early age. We conclude that Lrat-/- mice may serve as an animal model with early onset severe retinal dystrophy and severe retinyl ester deprivation.