Fatty acyl CoA-dependent and -independent retinol esterification by rat liver and lactating mammary gland microsomes

Retinol esterification was examined in microsomes from rat liver and lactating mammary gland as a function of the form of retinol substrate, dependence on fatty acyl CoA, and sensitivity to phenylmethylsulfonyl fluoride (PMSF). Retinol bound to cellular retinol-binding protein (CRBP) or dispersed in solvent was esterified in a fatty acyl CoA-independent, PMSF-sensitive reaction, consistent with lecithin:retinol acyltransferase (LRAT) activity. LRAT activity exhibited the same Km (2 microM retinol) between tissues but a higher Vmax in liver as compared to that in mammary gland (47 vs 8 pmol/min/mg microsome protein, respectively). Solvent-dispersed retinol was also esterified in a fatty acyl CoA-dependent, PMSF-resistant reaction, consistent with acyl CoA:retinol acyltransferase (ARAT) activity. Retinol bound to CRBP was not a good substrate for this reaction. ARAT activity displayed a similar Vmax (300 pmol/min/mg microsome protein) between tissues but Km values of 15 and 5 microM for retinol and fatty acyl CoA in mammary gland as compared to 30 and 25 microM, respectively, in the liver. Thus, when substrate was near or below Km, retinol esterification occurred predominantly by LRAT in the liver and ARAT in the mammary gland, respectively. The concentration of CRBP in the cytosol, determined by Western blotting, was approximately 2 microM in the liver but was almost nondetectable in the mammary gland. These data suggest that retinol esterification is regulated via different mechanisms in liver and mammary gland and support a specific role for CRBP in the liver.     

Acyl-CoA: retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT) activation during the lipocyte phenotype induction in hepatic stellate cells

We have examined retinol esterification in the established GRX cell line, representative of hepatic stellate cells, and in primary cultures of ex vivo purified murine hepatic stellate cells. The metabolism of [3H]retinol was compared in cells expressing the myofibroblast or the lipocyte phenotype, under the physiological retinol concentrations. Retinyl esters were the major metabolites, whose production was dependent upon both acyl-CoA:retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT). Lipocytes had a significantly higher esterification capacity than myofibroblasts. In order to distinguish the intrinsic enzyme activity from modulation of retinol uptake and CRBP-retinol content of the cytosol in the studied cells, we monitored enzyme kinetics in the purified microsomal fraction. We found that both LRAT and ARAT activities were induced during the conversion of myofibroblasts to lipocytes. LRAT induction was dependent upon retinoic acid, while that of ARAT was dependent upon the overall induction of the fat storing phenotype. The fatty acid composition of retinyl-esters suggested a preferential inclusion of exogenous fatty acids into retinyl esters. We conclude that both LRAT and ARAT participate in retinol esterification in hepatic stellate cells: LRAT's activity correlates with the vitamin A status, while ARAT depends upon the availability of fatty acyl-CoA and the overall lipid metabolism in hepatic stellate cells.     

Vitamin A metabolism in the human intestinal Caco-2 cell line

The human intestinal Caco-2 cell line, described as enterocyte-like in a number of studies, was examined for its ability to carry out the metabolism of vitamin A normally required in the absorptive process. Caco-2 cells contained cellular retinol-binding protein II, a protein which is abundant in human villus-associated enterocytes and may play an important role in the absorption of vitamin A. Microsomal preparations from Caco-2 cells contained retinal reductase, acyl-CoA-retinol acyltransferase (ARAT), and lecithin-retinol acyltransferase (LRAT) activities, which have previously been proposed to be involved in the metabolism of dietary vitamin A in the enterocyte. When intact Caco-2 cells were provided with beta-carotene, retinyl acetate, or retinol, synthesis of retinyl palmitoleate, oleate, palmitate, and small amounts of stearate resulted. However, exogenous retinyl palmitate or stearate was not used by Caco-2 cells as a source of retinol for ester synthesis. While there was a disproportionate synthesis of monoenoic fatty acid esters of retinol in Caco-2 cells compared to the retinyl esters typically found in human chylomicrons or the esters normally synthesized in rat intestine, the pattern was consistent with the substantial amount of unsaturated fatty acids, particularly 18:1 and 16:1, found in the sn-1 position of Caco-2 microsomal phosphatidylcholine, the fatty acyl donor for LRAT. Both ARAT and LRAT have been proposed to be responsible for retinyl ester synthesis in the enterocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

The molecular basis of retinoid absorption: a genetic dissection

The intestine and other tissues are able to synthesize retinyl esters in an acyl-CoA-dependent manner involving an acyl-CoA:retinol acyltransferase (ARAT). However, the molecular identity of this ARAT has not been established. Recent studies of lecithin:retinol acyltransferase (LRAT)-deficient mice indicate that LRAT is responsible for the preponderance of retinyl ester synthesis in the body, aside from in the intestine and adipose tissue. Our present studies, employing a number of mutant mouse models, identify diacylglycerol acyltransferase 1 (DGAT1) as an important intestinal ARAT in vivo. The contribution that DGAT1 makes to intestinal retinyl ester synthesis becomes greater when a large pharmacologic dose of retinol is administered by gavage to mice. Moreover, when large retinol doses are administered another intestinal enzyme(s) with ARAT activity becomes apparent. Surprisingly, although DGAT1 is expressed in adipose tissue, DGAT1 does not catalyze retinyl ester synthesis in adipose tissue in vivo. Our data also establish that cellular retinol-binding protein, type II (CRBPII), which is expressed solely in the adult intestine, in vivo channels retinol to LRAT for retinyl ester synthesis. Contrary to what has been proposed in the literature based on in vitro studies, CRBPII does not directly prevent retinol from being acted upon by DGAT1 or other intestinal ARATs in vivo.     

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.     

Esterification by rat liver microsomes of retinol bound to cellular retinol-binding protein

We have investigated the esterification by liver membranes of retinol bound to cellular retinol-binding protein (CRBP). When CRBP carrying [3H]retinol as its ligand was purified from rat liver cytosol and incubated with rat liver microsomes, a significant fraction of the [3H]retinol was converted to [3H]retinyl ester. Esterification of the CRBP-bound [3H]retinol, which was maximal at pH 6-7, did not require the addition of an exogenous fatty acyl group. Indeed, when additional palmitoyl-CoA or coenzyme A was provided, the rate of esterification increased either very slightly or not at all. The esterification reaction had a Km for [3H]retinol-CRBP of 4 +/- 0.6 microM and a maximum velocity of 145 +/- 52 pmol/min/mg of microsomal protein (n = 4). The major products were retinyl palmitate/oleate and retinyl stearate in a ratio of approximately 2 to 1 over a range of [3H]retinol-CRBP concentrations from 1 to 8 microM. The addition of progesterone, a known inhibitor of the acyl-CoA:retinol acyltransferase reaction, consistently increased the rate of retinyl ester formation when [3H]retinol was delivered bound to CRBP. These experiments indicate that retinol presented to liver microsomal membranes by CRBP can be converted to retinyl ester and that this process, in contrast to the esterification of dispersed retinol, is independent of the addition of an activated fatty acid and produces a pattern of retinyl ester species similar to that observed in intact liver. A possible role of phospholipids as endogenous acyl donors in the esterification of retinol bound to CRBP is supported by our observations that depletion of microsomal phospholipid with phospholipase A2 prior to addition of retinol-CRBP decreased the retinol-esterifying activity almost 50%. Conversely, incubating microsomes with a lipid-generating system containing choline, CDP-choline, glycerol 3-phosphate, and an acyl-CoA-generating system prior to addition of retinol-CRBP increased retinol esterification significantly as compared to buffer-treated controls.     

Esterification of retinol in rat liver. Possible participation by cellular retinol-binding protein and cellular retinol-binding protein II

Retinol bound to cellular retinol-binding protein (CRBP) was available for esterification by liver microsomes in the absence of exogenous acyl donors. Moreover, exogenous acyl-CoA gave little or no stimulation of ester production over what was observed with the endogenous acyl donor. In contrast, unbound retinol was esterified in an acyl-CoA-dependent reaction. The presence of two different enzyme activities, acyl-CoA-dependent and -independent, was demonstrated by differential sensitivities to several enzyme inhibitors. The enzyme reaction with retinol-CRBP and endogenous acyl donor produced retinyl esters normally found in vivo in liver. In addition, rates of esterification with this system were sufficient to maintain liver stores. Liver also contains cellular retinol-binding protein, type II (CRBP(II] during the perinatal period. Radioimmunoassay revealed highest levels of CRBP(II) in liver 3-4 days after birth. Examination of retinol esterification by microsomes from the liver of 3-day-old rats revealed a retinyl ester synthase activity with lower Km and higher Vmax than that found in the adult. The activity could use either retinol-CRBP or retinol-CRBP(II) and an endogenous acyl donor. The microsomes from 3-day-old liver had greater esterifying ability than microsomes from adult liver, perhaps due to the presence of two retinyl ester synthase enzymes.     

Acyl-CoA-independent esterification of retinol bound to cellular retinol-binding protein (type II) by microsomes from rat small intestine

Cellular retinol-binding protein (type II) (CRBP(II)), a newly described retinol-binding protein, is present in the small intestinal absorptive cell at high levels. Retinol (vitamin A alcohol) presented as a complex with CRBP(II) was found here to be esterified by microsomal preparations from rat small intestinal mucosa. The esterification observed utilized an endogenous acyl donor(s) and produced retinyl esters containing linoleate, oleate, palmitate, and stearate in a proportion quite similar to that previously reported for retinyl esters in lymph and isolated chylomicrons of rat. No dependence on endogenous or exogenous acyl-CoA could be demonstrated. The apparent Km for retinol-CRBP(II) in the reaction with endogenous acyl donor was 2.4 X 10(-7) M. Retinol presented as a complex with CRBP(II) was esterified more than retinol presented as a complex with cellular retinol-binding protein or retinol-binding protein, two other proteins known to bind retinol in vivo, but about the same as retinol presented bound to bovine serum albumin or beta-lactoglobulin. The ability of protein-bound retinol to be esterified was related to accessibility of the hydroxyl group, as judged by the ability of alcohol dehydrogenase to oxidize the bound retinol. However, whereas retinol bound to CRBP(II) was unavailable for esterification in any acyl-CoA-dependent reaction, retinol bound to bovine serum albumin was rapidly esterified in a reaction utilizing exogenous acyl-CoA. The results suggest that one of the functions of CRBP(II) is to accept retinol after it is absorbed or generated from carotenes in the small intestine and present it to the appropriate esterifying enzyme.     

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 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 lecithin:retinol acyltransferase activity in human and rat liver

This report demonstrates that exogenous phosphatidylcholine will serve as an acyl donor for the esterification of retinol complexed to cellular retinol-binding protein (CRBP) by human and rat liver microsomal preparations. The retinyl ester synthases utilized phosphatidylcholine but had little or no ability to transfer acyl groups from lysophosphatidylcholine, phosphatidyl-ethanolamine, or phosphatidic acid to retinol-CRBP. The human and rat activities also demonstrated positional selectivity as only the fatty acyl group at the sn-1 position of phosphatidylcholine was transferred. This in vitro activity may have considerable physiological importance since the fatty acyl composition at the sn-1 position of phosphatidylcholine is remarkably similar to the hepatic retinyl esters observed in vivo.     

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 μM and 13.9 ± 0.3 μM, 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 ～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 ～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.     

Lecithin:retinol acyltransferase in retinal pigment epithelial microsomes

Microsomal preparations from retinal pigment epithelium carry out phosphatidylcholine synthesis upon incubation with 1-palmitoyllysophosphatidylcholine and fatty acyl-CoA. Phosphatidylcholine synthesized in situ in this manner is an acyl donor for retinyl ester synthesis, demonstrating the existence of lecithin:retinol acyltransferase. Although acyl transfer to retinol is from the 1-position of phosphatidylcholine, the fatty acid in the 2-position is important in substrate recognition. The finding of this novel enzyme activity in retinal pigment epithelial microsomes suggests that phosphatidylcholine is the endogenous acyl donor in CoA-independent retinol esterification observed in these preparations.     

CoA- and non-CoA-dependent retinol esterification in retinal pigment epithelium

Washed, buffered microsomes from bovine retinal pigment epithelium catalyze retinyl ester synthesis from retinol in the absence of an exogenous acyl donor. A plot of retinyl ester synthesis versus time reaches a plateau at 123 +/- 26 nmol of retinyl ester mg-1 microsomal protein, providing a minimum value of the concentration of the endogenous acyl donor. Fatty acyl-CoA analysis by three different methods employing high performance liquid chromatography resulted in the detection of less than 1 nmol mg-1 protein of acyl-CoA, indicating that fatty acyl-CoA is not the endogenous acyl donor. Stimulation of the rate of retinyl ester synthesis by palmitoyl-CoA or ATP, CoA, and palmitate is observed following its addition at the beginning of the reaction or after the endogenous acyl source has been exhausted by 20 min of reaction with retinol. Palmitate from [14C]palmitoyl-CoA is incorporated into retinyl ester at a rate similar to that for the incorporation of [3H] retinol, demonstrating the presence of an apparent acyl-CoA:retinol acyl transferase activity. The acyl group from palmitoyl-CoA can be transferred initially to a component of the microsomes and subsequently to retinol. The product of retinyl ester synthesis from all-trans-retinol and palmitoyl-CoA is all-trans-retinyl palmitate, indicating that the stereochemical configuration is retained during esterification. The kinetic parameters for the esterification of 11-cis-retinol and all-trans-retinol are similar.     

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.     

Evidence for a lecithin-retinol acyltransferase activity in the rat small intestine

Cellular retinol-binding protein, type II (CRBP (II] is an abundant protein of the mature enterocytes of the small intestine. It has been shown to direct retinol to an acyl-CoA-independent esterifying activity that utilizes an endogenous acyl donor (Ong, D.E., Kakkad, B., and MacDonald, P.N. (1987) J. Biol. Chem. 262, 2729-2736). Here we report that this activity in intestinal microsomes will catalyze the transfer of acyl moieties from exogenous phosphatidylcholine (PC) to retinol-CRBP(II) to produce retinyl esters. The microsomal activity displayed positional selectivity as only the sn-1-acyl moiety of PC was transferred to retinol-CRBP(II). The retinyl ester synthase was selective for PC substrates as acyl transfer from phosphatidylethanolamine, phosphatidic acid, or free fatty acid to retinol-CRBP(II) was not observed. Some formation of retinyl esters was observed with exogenous acyl-CoA, but the amount produced was considerably lower than ester formation from exogenous PC and could be shown to be due to a different enzyme activity. Inhibitor studies clearly distinguished between the enzyme activities responsible for the acyl-CoA-dependent esterification and the phosphatidylcholine-dependent esterification of retinol. The results provide strong evidence that retinol-CRBP(II) esterification in the intestine proceeds via a phosphatidylcholine-dependent transacylase mechanism similar to that established for the esterification of cholesterol by lecithin-cholesterol acyltransferase.     

The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerols, waxes, and retinyl esters

The final step of triacylglycerol biosynthesis is catalyzed by acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes. The two known DGATs, DGAT1 and DGAT2, are encoded by unrelated genes. Although both DGAT1 and DGAT2 knockout mice have reduced tissue triacylglycerol contents, they have disparate phenotypes, prompting us to investigate whether the two enzymes have unrecognized functional differences. We now report that DGAT1 exhibits additional acyltransferase activities in vitro, including those of acyl CoA:monoacylglycerol acyltransferase (MGAT), wax monoester and wax diester synthases, and acyl CoA:retinol acyltransferase (ARAT), which catalyze the synthesis of diacylglycerols, wax esters, and retinyl esters, respectively. These activities were demonstrated in in vitro assays with membranes from insect cells or homogenates from COS7 cells overexpressing DGAT1. Wax synthase and ARAT activities were also demonstrated in intact COS7 cells expressing DGAT1. Additionally, cells and tissues from DGAT1-deficient mice exhibited reduced ARAT activity, and the mice had increased levels of unesterified retinol in their livers on a high-retinol diet. Our findings indicate that DGAT1 can utilize a variety of acyl acceptors as substrates in vitro and suggest that these activities may be relevant to the in vivo functions of DGAT1.     

Age-related variations in acyl-CoA:retinol acyltransferase activity and vitamin A concentration in the liver and epidermis of hairless mice

Since the factors regulating retinol esterification by acyl-CoA:retinol acyltransferase are poorly understood, we studied the age-related variations in acyl-CoA:retinol acyltransferase activity in hairless mice. Epidermis and liver were collected at intervals from birth to adolescence (0-6 weeks). Vitamin A was analyzed by high-performance liquid chromatography and acyl-CoA:retinol acyltransferase by an in vitro radioincubation assay of microsomes. Epidermal vitamin A (retinol plus retinyl esters) increased 8-10 times after birth and by the age of 3 weeks adult values were attained. This increase was accompanied by a 2-fold increase in acyl-CoA:retinol acyltransferase activity in the epidermis between 3 days and 6 weeks of age. In young animals the dependence of acyl-CoA:retinol acyltransferase on exogenous co-substrate (palmitoyl-CoA) was also lower than in adult animals. Although a pronounced age-related accumulation of retinol was recorded in the liver, the activity of acyl-CoA:retinol acyltransferase did not increase with age and there was no change in the dependence of acyl-CoA:retinol acyltransferase on exogenous palmitoyl-CoA.     

Retinyl esters are the substrate for isomerohydrolase

Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.