DGAT1: A Palmitoyl Coenzyme A-dependent Retinyl-Ester Synthase in Retina and RPE

Abstract

Purpose:: Regeneration of visual chromophore involves an enzyme pathway called the visual cycle. The retinoid isomerase in this pathway (Rpe65) converts a fatty-acyl ester of all-trans-retinol (all-trans-ROL) to 11-cis-retinol (11-cis-ROL). These all-trans-retinyl esters (all-trans-RE’s) are synthesized in the retinal pigment epithelium (RPE) by lecithin:retinol acyltransferase (LRAT). A second retinyl-ester synthase called acyl CoA:retinol acyltransferase (ARAT) has been reported in the RPE. Unlike LRAT, which uses phosphatidylcholine as a fatty-acyl donor, ARAT uses an activated fatty acid such as palmitoyl coenzyme A (palm CoA). The maximum turnover rate (V_max) of ARAT is similar to that of LRAT. However, the Michaelis constant (K_M) of ARAT for all-trans-ROL substrate is 10-fold higher than that of LRAT. These observations suggest that ARAT may complement LRAT to provide additional ester synthase capacity under conditions of high all-trans-ROL, such as following exposure to bright light. A strong candidate for ARAT is acyl CoA:diacylglyerol acyltransferase type-1 (DGAT1). In the current study we evaluated the role of DGAT1 in the processing of visual retinoids.

Methods:: We measured expression of the DGAT1 gene in chicken and bovine retinas and RPE using quantitative real-time PCR. We determined levels of endogenous retinoids in animal tissues by extracting tissue homogenates into hexane and analyzing with normal-phase liquid chromatography. To assay ARAT activity, we prepared homogenates from wild-type and dgat1 ^-/- knockout mouse retinas and RPE. These were incubated in assay mixtures containing all-trans-ROL plus or minus palm CoA. Reactions were quenched with methanol, extracted with hexane, and analyzed for production of all-trans-RE’s by liquid chromatography. ARAT activity was determined by measuring the difference between the rates of all-trans-RE synthesis in the absence and presence of palm CoA.

Results:: The mRNA for DGAT1 was expressed in both retina and RPE. Interestingly, the dgat1 mRNA was four-fold more abundant in cone-dominant chicken versus rod-dominant bovine retinas. Endogenous all-trans-RE’s and 11-cis-RE’s were less abundant in eyecups from light-adapted dgat1 ^-/- versus wild-type mice. Finally, ARAT catalytic activity was 10-fold decreased in RPE from dgat1 ^-/- versus wild-type mice.

Conclusions:: These observations indicate that DGAT1 accounts for at least a significant fraction of the observed ARAT activity in mouse retinas and RPE. It is less clear if DGAT1 represents the dominant palm CoA-dependent retinyl-ester synthase in the cone-dominant chicken retina.

Keywords: retinoids/retinoid binding proteins • retinal pigment epithelium • photoreceptors