Retinol Saturase Research Articles

Activation of Retinoic Acid Receptors by Dihydroretinoids

Vitamin A-derived metabolites act as ligands for nuclear receptors controlling the expression of a number of genes. Stereospecific saturation of the C(13)-C(14) double bond of all-trans-retinol by the enzyme, retinol saturase (RetSat), leads to the production of (R)-all-trans-13,14-dihydroretinol. In liver and adipose tissue, expression of RetSat is controlled by peroxisome proliferator-activated receptors (PPAR) alpha and gamma, respectively. Expression of RetSat in adipose tissue is also required for PPARgamma activation and adipocyte differentiation, but the involved mechanism is poorly understood. In this study, we examined the potential of (R)-all-trans-13,14-dihydroretinol and its metabolites to control gene transcription via nuclear receptors. Using a cell-based transactivation assay to screen 25 human nuclear receptors for activation, we found that dihydroretinoids have a narrow transcriptional profile limited primarily to activation of retinoic acid receptors (RARs). Although (R)-all-trans-13,14-dihydroretinoic acid exhibited comparable potency to retinoic acid in promoting the interaction of RARs with a coactivator peptide in vitro, its potency in activating RAR-controlled genes in cell-based assays was much lower than that of retinoic acid. As an explanation for the weak RAR agonist activity of dihydroretinoids in cell-based assays, we propose that both delivery of ligand to the nucleus and RAR activation favor retinoic acid over dihydroretinoids. Discrimination between the cognate ligand, retinoic acid, and close analogs such as dihydroretinoids, occurs at multiple levels and may represent a mechanism to modulate retinoid-dependent physiological processes.

Correction for Schupp et al., Retinol saturase promotes adipogenesis and is downregulated in obesity

Correction for Schupp et al., Retinol saturase promotes adipogenesis and is downregulated in obesity

Retinol saturase promotes adipogenesis and is downregulated in obesity

Adipocyte differentiation is controlled by many transcription factors, but few known downstream targets of these factors are necessary for adipogenesis. Here we report that retinol saturase (RetSat), which is an enzyme implicated in the generation of dihydroretinoid metabolites, is induced during adipogenesis and is directly regulated by the transcription factor peroxisome proliferator activated receptor gamma (PPARgamma). Ablation of RetSat dramatically inhibited adipogenesis but, surprisingly, this block was not overcome by the putative product of RetSat enzymatic activity. On the other hand, ectopic RetSat with an intact, but not a mutated, FAD/NAD dinucleotide-binding motif increased endogenous PPARgamma transcriptional activity and promoted adipogenesis. Indeed, RetSat was not required for adipogenesis when cells were provided with exogenous PPARgamma ligands. In adipose tissue, RetSat is expressed in adipocytes but is unexpectedly downregulated in obesity, most likely owing to infiltration of macrophages that we demonstrate to repress RetSat expression. Thiazolidinedione treatment reversed low RetSat expression in adipose tissue of obese mice. Thus, RetSat plays an important role in the biology of adipocytes, where it favors normal differentiation, yet is reduced in the obese state. RetSat is thus a novel target for therapeutic intervention in metabolic disease.

Iron supplementation modulates genes involved in oxidoreductase activity, including prostaglandin‐endoperoxide synthase 2 in intestinal tumors

To determine the effect of excess dietary iron on gene expression of intestinal tumors in an animal model of human familial adenomatous polyposis, male C57BL/6J ApcMin/+ mice were fed adequate and excess amounts of iron (ferrous sulfate: 45 vs. 450 mg iron/kg AIN‐93M[M] diet). After 10 wk, mice were sacrificed and intestines removed. Tumors and adjacent non‐tumor tissue were excised and RNA isolated for micro‐array analysis (n=3/group; Mouse Genome 430A 2.0 arrays; Affymetrix) and RT‐PCR. Pathway mapping was performed to reveal gene networks (Pathway Studio 4.0, Ariadne Genomics, Inc.; GeneSpring, Agilent, Inc.; Searcher, Lucidyx). Bayesian ANOVA was used for micro‐arrays. Data show that intestinal tumors alter expression of a group of genes involved in oxidoreductase activity when exposed to excess iron. Differentially expressed genes include prostaglandin‐endoperoxide synthase 2, retinol saturase, alcohol dehydrogenase, deiodinase, cytochrome P450 (family 2, 4), and glutathione peroxidase. Findings reveal that iron supplementation modulates oxidoreductase activity of intestinal tumors, possibly in response to the pro‐oxidative, pro‐inflammatory effects of excess intraluminal iron. Supported in part by grants IRG‐91‐022 from ACS (to JS) and P30CA43703 from NIH. Ferrous sulfate provided by Crown Technologies, Inc.

Stereospecificity of retinol saturase: absolute configuration, synthesis, and biological evaluation of dihydroretinoids.

Retinol saturase carries out a stereospecific saturation of the C13−C14 double bond of all-trans-retinol to generate (13R)-all-trans-13,14-dihydroretinol. This compound is found in cells expressing mouse or zebrafish retinol saturase and in the livers of mice fed retinyl palmitate. All-trans-13,14-dihydroretinol is oxidized in vivo to all-trans-13,14-dihydroretinoic acid, a highly selective agonist of the retinoic acid receptor. The naturally occurring (13R)-all-trans-13,14-dihydroretinoic acid is a weaker agonist than the (13S) enantiomer, indicating enantioselective recognition by the ligand-binding pocket of this receptor. Consequently the (13S) enantiomer, acting through the retinoic acid receptor, also inhibits adipose differentiation more potently than the (13R) enantiomer.

An RNAi-based genetic screen for oxidative stress resistance reveals retinol saturase as a mediator of stress resistance

Oxidative stress has been implicated in the pathogenesis of numerous late-onset diseases as well as organismal longevity. Nevertheless, the genetic components that affect cellular sensitivity to oxidative stress have not been explored extensively at the genome-wide level in mammals. Here we report an RNA interference (RNAi) screen for genes that increase resistance to an organic oxidant, tert-butylhydroperoxide ( tert-BHP), in cultured fibroblasts. The loss-of-function screen allowed us to identify several short hairpin RNAs (shRNAs) that elevated the cellular resistance to tert-BHP. One of these shRNAs strongly protected cells from tert-BHP and H 2O 2 by specifically reducing the expression of retinol saturase, an enzyme that converts all- trans-retinol (vitamin A) to all- trans-13,14-dihydroretinol. The protective effect was well correlated with the reduction in mRNA level and was observed in both primary fibroblasts and NIH3T3 cells. The results suggest a novel role for retinol saturase in regulating sensitivity to oxidative stress and demonstrate the usefulness of large-scale RNAi screening for elucidating new molecular pathways involved in stress resistance.

Specificity of Zebrafish Retinol Saturase: Formation of All-trans-13,14-dihydroretinol and All-trans-7,8- dihydroretinol

Metabolism of vitamin A, all-trans-retinol, leads to the formation of 11-cis-retinaldehyde, the visual chromophore, and all-trans-retinoic acid, which is involved in the regulation of gene expression through the retinoic acid receptor. Enzymes and binding proteins involved in retinoid metabolism are highly conserved across species. We previously described a novel mammalian enzyme that saturates the 13-14 double bond of all-trans-retinol to produce all-trans-13,14-dihydroretinol, which then follows the same metabolic fate as that of all-trans-retinol. Specifically, all-trans-13,14-dihydroretinol is transiently oxidized to all-trans-13,14-dihydroretinoic acid before being oxidized further by Cyp26 enzymes. Here, we report the identification of two putative RetSat homologues in zebrafish, one of which, zebrafish RetSat A (zRetSat A), also had retinol saturase activity, whereas zebrafish RetSat B (zRetSat B) was inactive under similar conditions. Unlike mouse RetSat (mRetSat), zRetSat A had an altered bond specificity saturating either the 13-14 or 7-8 double bonds of all-trans-retinol to produce either all-trans-13,14-dihydroretinol or all-trans-7,8-dihydroretinol, respectively. zRetSat A also saturated the 13-14 or 7-8 double bonds of all-trans-3,4-didehydroretinol (vitamin A2), a second endogenous form of vitamin A in zebrafish. The dual enzymatic activity of zRetSat A displays a newly acquired specificity for the 13-14 double bond retained in higher vertebrates and also the evolutionarily preserved activity of bacterial phytoene desaturases and plant carotenoid isomerases. Expression of zRetSat A was restricted to the liver and intestine of hatchlings and adult zebrafish, whereas zRetSat B was expressed in the same tissues but at earlier developmental stages. Exogenous all-trans-retinol, all-trans-13,14-dihydroretinol, or all-trans-7,8-dihydroretinol led to the strong induction of the expression of the retinoic acid-metabolizing enzyme, Cyp26A1, arguing for an active signaling function of dihydroretinoid metabolites in zebrafish. These findings point to a conserved function but altered specificity of RetSat in vertebrates, leading to the generation of various dihydroretinoid compounds, some of which could have signaling functions.

Metabolism and Transactivation Activity of 13,14-Dihydroretinoic Acid

The metabolism of vitamin A is a highly regulated process that generates essential mediators involved in the development, cellular differentiation, immunity, and vision of vertebrates. Retinol saturase converts all-trans-retinol to all-trans-13,14-dihydroretinol (Moise, A. R., Kuksa, V., Imanishi, Y., and Palczewski, K. (2004) J. Biol. Chem. 279, 50230-50242). Here we demonstrate that the enzymes involved in oxidation of retinol to retinoic acid and then to oxidized retinoic acid metabolites are also involved in the synthesis and oxidation of all-trans-13,14-dihydroretinoic acid. All-trans-13,14-dihydroretinoic acid can activate retinoic acid receptor/retinoid X receptor heterodimers but not retinoid X receptor homodimers in reporter cell assays. All-trans-13,14-dihydroretinoic acid was detected in vivo in Lrat-/- mice supplemented with retinyl palmitate. Thus, all-trans-13,14-dihydroretinoic acid is a naturally occurring retinoid and a potential ligand for nuclear receptors. This new metabolite can also be an intermediate in a retinol degradation pathway or it can serve as a precursor for the synthesis of bioactive 13,14-dihydroretinoid metabolites.

Identification of All-trans-Retinol:All-trans-13,14-dihydroretinol Saturase

Retinoids carry out essential functions in vertebrate development and vision. Many of the retinoid processing enzymes remain to be identified at the molecular level. To expand the knowledge of retinoid biochemistry in vertebrates, we studied the enzymes involved in plant metabolism of carotenoids, a related group of compounds. We identified a family of vertebrate enzymes that share significant similarity and a putative phytoene desaturase domain with a recently described plant carotenoid isomerase (CRTISO), which isomerizes prolycopene to all-trans-lycopene. Comparison of heterologously expressed mouse and plant enzymes indicates that unlike plant CRTISO, the CRTISO-related mouse enzyme is inactive toward prolycopene. Instead, the CRTISO-related mouse enzyme is a retinol saturase carrying out the saturation of the 13-14 double bond of all-trans-retinol to produce all-trans-13,14-dihydroretinol. The product of mouse retinol saturase (RetSat) has a shifted UV absorbance maximum, lambda(max) = 290 nm, compared with the parent compound, all-trans-retinol (lambda(max) = 325 nm), and its MS analysis (m/z = 288) indicates saturation of a double bond. The product was further identified as all-trans-13,14-dihydroretinol, since its characteristics were identical to those of a synthetic standard. Mouse RetSat is membrane-associated and expressed in many tissues, with the highest levels in liver, kidney, and intestine. All-trans-13,14-dihydroretinol was also detected in several tissues of animals maintained on a normal diet. Thus, saturation of all-trans-retinol to all-trans-13,14-dihydroretinol by RetSat produces a new metabolite of yet unknown biological function.