Modest Decreases in Endogenous All-trans-Retinoic Acid Produced by a Mouse Rdh10 Heterozygote Provoke Major Abnormalities in Adipogenesis and Lipid Metabolism.
ABSTRACT: Pharmacological dosing of all-trans-retinoic acid (atRA) controls adiposity in rodents by inhibiting adipogenesis and inducing fatty acid oxidation. Retinol dehydrogenases (Rdh) catalyze the first reaction that activates retinol into atRA. This study examined postnatal contributions of Rdh10 to atRA biosynthesis and physiological functions of endogenous atRA. Embryonic fibroblasts from Rdh10 heterozygote hypomorphs or with a total Rdh10 knockout exhibit decreased atRA biosynthesis and escalated adipogenesis. atRA or a retinoic acid receptor (RAR) pan-agonist reversed the phenotype. Eliminating one Rdh10 copy in vivo (Rdh10+/- ) yielded a modest decrease (≤25%) in the atRA concentration of liver and adipose but increased adiposity in male and female mice fed a high-fat diet (HFD); increased liver steatosis, glucose intolerance, and insulin resistance in males fed an HFD; and activated bone marrow adipocyte formation in females, regardless of dietary fat. Chronic dosing with low-dose atRA corrected the metabolic defects. These data resolve physiological actions of endogenous atRA, reveal sex-specific effects of atRA in vivo, and establish the importance of Rdh10 to metabolic control by atRA. The consequences of a modest decrease in tissue atRA suggest that impaired retinol activation may contribute to diabesity, and low-dose atRA therapy may ameliorate adiposity and its sequelae of glucose intolerance and insulin resistance.
Project description:Retinoic acid (atRA) signaling is essential for regulating embryonic development, and atRA levels must be tightly controlled in order to prevent congenital abnormalities and fetal death which can result from both excessive and insufficient atRA signaling. Cellular enzymes synthesize atRA from Vitamin A, which is obtained from dietary sources. Embryos express multiple enzymes that are biochemically capable of catalyzing the initial step of Vitamin A oxidation, but the precise contribution of these enzymes to embryonic atRA synthesis remains unknown. Using Rdh10(trex)-mutant embryos, dietary supplementation of retinaldehyde, and retinol dehydrogenase (RDH) activity assays, we demonstrate that RDH10 is the primary RDH responsible for the first step of embryonic Vitamin A oxidation. Moreover, we show that this initial step of atRA synthesis occurs predominantly in a membrane-bound cellular compartment, which prevents inhibition by the cytosolic cellular retinol-binding protein (RBP1). These studies reveal that widely expressed cytosolic enzymes with RDH activity play a very limited role in embryonic atRA synthesis under normal dietary conditions. This provides a breakthrough in understanding the precise cellular mechanisms that regulate Vitamin A metabolism and the synthesis of the essential embryonic regulatory molecule atRA.
Project description:Human retinol dehydrogenase 10 (RDH10) was implicated in the oxidation of all-trans-retinol for biosynthesis of all-trans-retinoic acid, however, initial assays suggested that RDH10 prefers NADP(+) as a cofactor, undermining its role as an oxidative enzyme. Here, we present evidence that RDH10 is, in fact, a strictly NAD(+)-dependent enzyme with multisubstrate specificity that recognizes cis-retinols as well as all-trans-retinol as substrates. RDH10 has a relatively high apparent K(m) value for NAD(+) (~100 microm) but the lowest apparent K(m) value for all-trans-retinol (~0.035 microm) among all NAD(+)-dependent retinoid oxidoreductases. Due to its high affinity for all-trans-retinol, RDH10 exhibits a greater rate of retinol oxidation in the presence of cellular retinol-binding protein type I (CRBPI) than human microsomal RoDH4, but like RoDH4, RDH10 does not recognize retinol bound to CRBPI as a substrate. Consistent with its preference for NAD(+), RDH10 functions exclusively in the oxidative direction in the cells, increasing the levels of retinaldehyde and retinoic acid. Targeted small interfering RNA-mediated silencing of endogenous RDH10 or RoDH4 expression in human cells results in a significant decrease in retinoic acid production from retinol, identifying both human enzymes as physiologically relevant retinol dehydrogenases. The dual cis/trans substrate specificity suggests a dual physiological role for RDH10: in the biosynthesis of 11-cis-retinaldehyde for vision as well as the biosynthesis of all-trans-retinoic acid for differentiation and development.
Project description:All-trans retinoic acid (ATRA) has a key role in dendritic cells (DCs) and affects T cell subtype specification and gut homing. However, the identity of the permissive cell types and the required steps of conversion of vitamin A to biologically active ATRA bringing about retinoic acid receptor-regulated signaling remains elusive. Here we present that only a subset of murine and human DCs express the necessary enzymes, including RDH10, RALDH2, and transporter cellular retinoic acid binding protein (CRABP)2, to produce ATRA and efficient signaling. These permissive cell types include CD103(+) DCs, granulocyte-macrophage colony-stimulating factor, and interleukin-4-treated bone marrow-derived murine DCs and human monocyte-derived DCs (mo-DCs). Importantly, in addition to RDH10 and RALDH2, CRABP2 also appears to be regulated by the fatty acid-sensing nuclear receptor peroxisome proliferator-activated receptor ? (PPAR?) and colocalize in human gut-associated lymphoid tissue DCs. In our model of human mo-DCs, all three proteins (RDH10, RALDH2, and CRABP2) appeared to be required for ATRA production induced by activation of PPAR? and therefore form a linear pathway. This now functionally validated PPAR?-regulated ATRA producing and signaling axis equips the cells with the capacity to convert precursors to active retinoids in response to receptor-activating fatty acids and is potentially amenable to intervention in diseases involving or affecting mucosal immunity.
Project description:Regulation of patterning and morphogenesis during embryonic development depends on tissue-specific signaling by retinoic acid (RA), the active form of Vitamin A (retinol). The first enzymatic step in RA synthesis, the oxidation of retinol to retinal, is thought to be carried out by the ubiquitous or overlapping activities of redundant alcohol dehydrogenases. The second oxidation step, the conversion of retinal to RA, is performed by retinaldehyde dehydrogenases. Thus, the specific spatiotemporal distribution of retinoid synthesis is believed to be controlled exclusively at the level of the second oxidation reaction. In an N-ethyl-N-nitrosourea (ENU)-induced forward genetic screen we discovered a new midgestation lethal mouse mutant, called trex, which displays craniofacial, limb, and organ abnormalities. The trex phenotype is caused by a mutation in the short-chain dehydrogenase/reductase, RDH10. Using protein modeling, enzymatic assays, and mutant embryos, we determined that RDH10(trex) mutant protein lacks the ability to oxidize retinol to retinal, resulting in insufficient RA signaling. Thus, we show that the first oxidative step of Vitamin A metabolism, which is catalyzed in large part by the retinol dehydrogenase RDH10, is critical for the spatiotemporal synthesis of RA. Furthermore, these results identify a new nodal point in RA metabolism during embryogenesis.
Project description:All-trans-retinoic acid (atRA) stimulates neurogenesis, dendritic growth of hippocampal neurons, and higher cognitive functions, such as spatial learning and memory formation. Although astrocyte-derived atRA has been considered a key factor in neurogenesis, little direct evidence identifies hippocampus cell types and the enzymes that biosynthesize atRA. Here we show that primary rat astrocytes, but not neurons, biosynthesize atRA using multiple retinol dehydrogenases (Rdh) of the short chain dehydrogenase/reductase gene family and retinaldehyde dehydrogenases (Raldh). Astrocytes secrete atRA into their medium; neurons sequester atRA. The first step, conversion of retinol into retinal, is rate-limiting. Neurons and astrocytes both synthesize retinyl esters and reduce retinal into retinol. siRNA knockdown indicates that Rdh10, Rdh2 (mRdh1), and Raldh1, -2, and -3 contribute to atRA production. Knockdown of the Rdh Dhrs9 increased atRA synthesis ?40% by increasing Raldh1 expression. Immunocytochemistry revealed cytosolic and nuclear expression of Raldh1 and cytosol and perinuclear expression of Raldh2. atRA autoregulated its concentrations by inducing retinyl ester synthesis via lecithin:retinol acyltransferase and stimulating its catabolism via inducing Cyp26B1. These data show that adult hippocampus astrocytes rely on multiple Rdh and Raldh to provide a paracrine source of atRA to neurons, and atRA regulates its own biosynthesis in astrocytes by directing flux of retinol. Observation of cross-talk between Dhrs9 and Raldh1 provides a novel mechanism of regulating atRA biosynthesis.
Project description:In mammals, the epithelial tissues of major salivary glands generate saliva and drain it into the oral cavity. For submandibular salivary glands (SMGs), the epithelial tissues arise during embryogenesis from naïve oral ectoderm adjacent to the base of the tongue, which begins to thicken, express SOX9 and invaginate into underlying mesenchyme. The developmental mechanisms initiating salivary gland development remain unexplored. In this study, we show that retinoic acid (RA) signaling activity at the site of gland initiation is colocalized with expression of retinol metabolic genes Rdh10 and Aldh1a2 in the underlying SMG mesenchyme. Utilizing a novel ex vivo assay for SMG initiation developed for this study, we show that RDH10 and RA are required for salivary gland initiation. Moreover, we show that the requirement for RA in gland initiation involves canonical signaling through retinoic acid receptors (RAR). Finally, we show that RA signaling essential for gland initiation is transduced specifically through RARα, with no contribution from other RAR isoforms. This is the first study to identify a molecular signal regulating mammalian salivary gland initiation.
Project description:The retinoic acid-inducible dehydrogenase reductase 3 (DHRS3) is thought to function as a retinaldehyde reductase that controls the levels of all-trans-retinaldehyde, the immediate precursor for bioactive all-trans-retinoic acid. However, the weak catalytic activity of DHRS3 and the lack of changes in retinaldehyde conversion to retinol and retinoic acid in the cells overexpressing DHRS3 undermine its role as a physiologically important all-trans-retinaldehyde reductase. This study demonstrates that DHRS3 requires the presence of retinol dehydrogenase 10 (RDH10) to display its full catalytic activity. The RDH10-activated DHRS3 acts as a robust high affinity all-trans-retinaldehyde-specific reductase that effectively converts retinaldehyde back to retinol, decreasing the rate of retinoic acid biosynthesis. In turn, the retinol dehydrogenase activity of RDH10 is reciprocally activated by DHRS3. At E13.5, DHRS3-null embryos have ?4-fold lower levels of retinol and retinyl esters, but only slightly elevated levels of retinoic acid. The membrane-associated retinaldehyde reductase and retinol dehydrogenase activities are decreased by ?4- and ?2-fold, respectively, in Dhrs3(-/-) embryos, and Dhrs3(-/-) mouse embryonic fibroblasts exhibit reduced metabolism of both retinaldehyde and retinol. Neither RDH10 nor DHRS3 has to be itself catalytically active to activate each other. The transcripts encoding DHRS3 and RDH10 are co-localized at least in some tissues during development. The mutually activating interaction between the two related proteins may represent a highly sensitive and conserved mechanism for precise control over the rate of retinoic acid biosynthesis.
Project description:RDH1 is one of the several enzymes that catalyze the first of the two reactions to convert retinol into all-trans-retinoic acid (atRA). Here, we show that Rdh1-null mice fed a low-fat diet gain more weight as adiposity (17% males, 13% females) than wild-type mice by 20 weeks old, despite neither consuming more calories nor decreasing activity. Glucose intolerance and insulin resistance develop following increased adiposity. Despite the increase in white fat pads, epididymal white adipose does not express Rdh1, nor does muscle. Brown adipose tissue (BAT) and liver express Rdh1 at relatively high levels compared to other tissues. Rdh1 ablation lowered body temperatures during ambient conditions. Given the decreased body temperature, we focused on BAT. A lack of differences in BAT adipogenic gene expression between Rdh1-null mice and wild-type mice, including Pparg, Prdm16, Zfp516 and Zfp521, indicated that the phenotype was not driven by brown adipose hyperplasia. Rather, Rdh1 ablation eliminated the increase in BAT atRA that occurs after re-feeding. This disruption of atRA homeostasis increased fatty acid uptake, but attenuated lipolysis in primary brown adipocytes, resulting in increased lipid content and larger lipid droplets. Rdh1 ablation also decreased mitochondrial proteins, including CYCS and UCP1, the mitochondria oxygen consumption rate, and disrupted the mitochondria membrane potential, further reflecting impaired BAT function, resulting in both BAT and white adipose hypertrophy. RNAseq revealed dysregulation of 424 BAT genes in null mice, which segregated predominantly into differences after fasting vs after re-feeding. Exceptions were Rbp4 and Gbp2b, which increased during both dietary conditions. Rbp4 encodes the serum retinol-binding protein-an insulin desensitizer. Gbp2b encodes a GTPase. Because Gbp2b increased several hundred-fold, we overexpressed it in brown adipocytes. This caused a shift to larger lipid droplets, suggesting that GBP2b affects signaling downstream of the ?-adrenergic receptor during basal thermogenesis. Thus, Rdh1-generated atRA in BAT regulates multiple genes that promote BAT adaptation to whole-body energy status, such as fasting and re-feeding. These gene expression changes promote optimum mitochondria function and thermogenesis, limiting adiposity. Attenuation of adiposity and insulin resistance suggests that RDH1 mitigates metabolic syndrome.
Project description:Craniofacial development is a complex process that involves sequential growth and fusion of the facial prominences. When these processes fail, congenital craniofacial anomalies can occur. For example, choanal atresia (CA) is a congenital craniofacial anomaly in which the connection between the nasal airway and nasopharynx is completely blocked. CA occurs in approximately 1/5000 live births and is a frequent component of congenital disorders such as CHARGE, Treacher Collins, Crouzon and Pfeiffer syndromes. However, the detailed cellular and molecular mechanisms underpinning the etiology and pathogenesis of CA remain elusive. In this study, we discovered that mice with mutations in retinol dehydrogenase 10 (Rdh10), which perturbs Vitamin A metabolism and retinoid signaling, exhibit fully penetrant CA. Interestingly, we demonstrate Rdh10 is specifically required in non-neural crest cells prior to E10.5 for proper choanae formation, and that in the absence of Rdh10, Fgf8 is ectopically expressed in the nasal fin. Furthermore, we found that defects in choanae development are associated with decreased cell proliferation and increased cell death in the epithelium of the developing nasal cavity, which retards invagination of the nasal cavity, and thus appears to contribute to the pathogenesis of CA. Taken together, our findings demonstrate that RDH10 is essential during the early stages of facial morphogenesis for the formation of a functional nasal airway, and furthermore establish Rdh10 mutant mice as an important model system to study CA.
Project description:All-trans-retinoic acid (RA) is a bioactive derivative of vitamin A that serves as an activating ligand for nuclear transcription factors, retinoic acid receptors. RA biosynthesis is initiated by the enzymes that oxidize retinol to retinaldehyde. It is well established that retinol dehydrogenase 10 (RDH10, SDR16C4), which belongs to the 16C family of the short chain dehydrogenase/reductase (SDR) superfamily of proteins, is the major enzyme responsible for the oxidation of retinol to retinaldehyde for RA biosynthesis during embryogenesis. However, several lines of evidence point towards the existence of additional retinol dehydrogenases that contribute to RA biosynthesis in vivo. In close proximity to RDH10 gene on human chromosome 8 are located two genes that are phylogenetically related to RDH10. The predicted protein products of these genes, retinol dehydrogenase epidermal 2 (RDHE2, SDR16C5) and retinol dehydrogenase epidermal 2-similar (RDHE2S, SDR16C6), share 59% and 56% sequence similarity with RDH10, respectively. Previously, we showed that the single ortholog of the human RDHE2 and RDHE2S in frogs, Xenopus laevis rdhe2, oxidizes retinol to retinaldehyde and is essential for frog embryonic development. In this study, we explored the potential of each of the two human proteins to contribute to RA biosynthesis. The results of this study demonstrate that human RDHE2 exhibits a relatively low but reproducible activity when expressed in either HepG2 or HEK293 cells. Expression of the native RDHE2 is downregulated in the presence of elevated levels of RA. On the other hand, the protein encoded by the human RDHE2S gene is unstable when expressed in HEK293 cells. RDHE2S protein produced in Sf9 cells is stable but has no detectable catalytic activity towards retinol. We conclude that the human RDHE2S does not contribute to RA biosynthesis, whereas the low-activity RA-sensitive human RDHE2 may have a role in adjusting the cellular levels of RA in accord with specific physiological conditions.