Youn Kyung Kim

Retinyl Ester Formation by Lecithin:Retinol Acyltransferase Is a Key Regulator of Retinoid Homeostasis in Mouse Embryogenesis

The developing mammalian embryo is entirely dependent on the maternal circulation for its supply of retinoids (vitamin A and its metabolites). The mechanisms through which mammalian developing tissues maintain adequate retinoid levels in the face of suboptimal or excessive maternal dietary vitamin A intake have not been established. We investigated the role of retinyl ester formation catalyzed by lecithin:retinol acyltransferase (LRAT) in regulating retinoid homeostasis during embryogenesis. Dams lacking both LRAT and retinol-binding protein (RBP), the sole specific carrier for retinol in serum, were maintained on diets containing different amounts of vitamin A during pregnancy. We hypothesized that the lack of both proteins would make the embryo more vulnerable to changes in maternal dietary vitamin A intake. Our data demonstrate that maternal dietary vitamin A deprivation during pregnancy generates a severe retinoid-deficient phenotype of the embryo due to the severe retinoid-deficient status of the double mutant dams rather than to the lack of LRAT in the developing tissues. Moreover, in the case of excessive maternal dietary vitamin A intake, LRAT acts together with Cyp26A1, one of the enzymes that catalyze the degradation of retinoic acid, and possibly with STRA6, the recently identified cell surface receptor for retinol-RBP, in maintaining adequate levels of retinoids in embryonic and extraembryonic tissues. In contrast, the pathway of retinoic acid synthesis does not contribute significantly to regulating retinoid homeostasis during mammalian development except under conditions of severe maternal retinoid deficiency.

Maternal-fetal transfer and metabolism of vitamin A and its precursor β-carotene in the developing tissues

The requirement of the developing mammalian embryo for retinoic acid is well established. Retinoic acid, the active form of vitamin A, can be generated from retinol and retinyl ester obtained from food of animal origin, and from carotenoids, mainly β-carotene, from vegetables and fruits. The mammalian embryo relies on retinol, retinyl ester and β-carotene circulating in the maternal bloodstream for its supply of vitamin A. The maternal-fetal transfer of retinoids and carotenoids, as well as the metabolism of these compounds in the developing tissues are still poorly understood. The existing knowledge in this field has been summarized in this review in reference to our basic understanding of the transport and metabolism of retinoids and carotenoids in adult tissues. The need for future research on the metabolism of these essential lipophilic nutrients during development is highlighted. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.

β-Carotene and its cleavage enzyme β-carotene-15,15`-oxygenase (CMOI) affect retinoid metabolism in developing tissues

The mammalian embryo relies on maternal circulating retinoids (vitamin A derivatives) for development. β‐Carotene is the major human dietary provitamin A. β‐Carotene‐15,15′‐oxygenase (CMOI) has been proposed as the main enzyme generating retinoid from β‐carotene in vivo. CMOI is expressed in embryonic tissues, suggesting that β‐carotene provides retinoids locally during development. We performed loss of CMOI function studies in mice lacking retinol‐binding protein (RBP), an established model of embryonic vitamin A deficiency (VAD). We show that, unexpectedly, lack of CMOI in the developing tissues further exacerbates the severity of VAD and thus the embryonic malformations of RBP−/− mice. Since β ‐carotene was not present in any of the mouse diets, we unveiled a novel action of CMOI independent from its β ‐carotene cleavage activity. We also show for the first time that CMOI exerts an additional function on retinoid metabolism by influencing retinyl ester formation via modulation of lecithin:retinol acyltransferase (LRAT) activity, at least in developing tissues. Finally, we demonstrate unequivocally that β‐carotene can serve as an alternative vitamin A source for the in situ synthesis of retinoids in developing tissues by the action of CMOI.—Kim, Y.‐K., Wassef, L., Chung, S., Jiang, H., Wyss, A., Blaner, W. S., Quadro, L. β‐Carotene and its cleavage enzyme β‐carotene‐15,15′‐oxygenase (CMOI) affect retinoid metabolism in developing tissues. FASEB J. 25, 1641–1652 (2011). www.fasebj.org

Prenatal retinoid deficiency leads to airway hyperresponsiveness in adult mice

There is increasing evidence that vitamin A deficiency in utero correlates with abnormal airway smooth muscle (SM) function in postnatal life. The bioactive vitamin A metabolite retinoic acid (RA) is essential for formation of the lung primordium; however, little is known about the impact of early fetal RA deficiency on postnatal lung structure and function. Here, we provide evidence that during murine lung development, endogenous RA has a key role in restricting the airway SM differentiation program during airway formation. Using murine models of pharmacological, genetic, and dietary vitamin A/RA deficiency, we found that disruption of RA signaling during embryonic development consistently resulted in an altered airway SM phenotype with markedly increased expression of SM markers. The aberrant phenotype persisted postnatally regardless of the adult vitamin A status and manifested as structural changes in the bronchial SM and hyperresponsiveness of the airway without evidence of inflammation. Our data reveal a role for endogenous RA signaling in restricting SM differentiation and preventing precocious and excessive SM differentiation when airways are forming.

Reverse-phase high-performance liquid chromatography (HPLC) analysis of retinol and retinyl esters in mouse serum and tissues.

The ability to measure endogenous metabolites of retinoids (vitamin A and its derivatives) in biological samples is key to understanding the crucial physiological actions of vitamin A. Over the years, many assays and methods have been developed to analyze different retinoids in biological samples. Liquid chromatography is the best analytical technique for routine analysis of these compounds. However, due to their different chemical properties, different retinoid metabolites cannot be accurately separated and quantified in a single chromatographic run. Here, we will describe a reverse-phase HPLC isocratic method that enables extraction, separation, identification, and quantification of all-trans-retinol and different molecular species of retinyl ester with high accuracy, sensitivity, and reliability.

Structure of the STRA6 receptor for retinol uptake.

A window into the cell for vitamin A Vitamin A is an essential nutrient for mammals, and its metabolites affect diverse biological processes. It is carried in the bloodstream as retinol by retinol binding protein (RBP); a protein called STRA6 is implicated in facilitating retinol translocation across the cell membrane. Chen et al. determined the structure of zebrafish STRA6 to a resolution of 3.9 Å by electron microscopy. A lipophilic cleft is a likely binding site for RBP, and an opening in the cleft may allow retinol to diffuse into the membrane. Unexpectedly, the structure also includes bound calcium-modulated protein, but its function remains unclear. Science, this issue p. 887 The structure of a STRA6-calmodulin complex gives insight into how retinol (vitamin A) enters cells. INTRODUCTION Vitamin A is an essential nutrient for all mammals, being vital for vision and for transcription of a wide array of genes. Retinol (vitamin A alcohol) is the predominant circulating retinoid. In the fasting state, retinol from liver stores is mobilized bound to retinol-binding protein (RBP), which transports this highly hydrophobic molecule in the bloodstream. How retinol is released from RBP and internalized by target cells has been the subject of intense debate. The RBP receptor, STRA6, was cloned in 2007. STRA6 was predicted to be a 75-kDa multipass transmembrane (TM) protein without sequence similarity to any known transporter, channel, or receptor. STRA6 is expressed widely, with particular abundance in the eye and placenta. Mutations in the human STRA6 gene have been linked to Matthew-Wood syndrome, which presents with ocular abnormalities and developmental defects. RATIONALE Despite a wealth of biochemical work aimed at investigating how STRA6 mediates internalization of retinol from RBP, progress at the molecular level has been hindered by the absence of structural information. Purified STRA6 from zebrafish was a detergent-stable dimer in an unexpected association with calmodulin (CaM), forming a 180-kDa complex. RESULTS Using cryo-electron microscopy, we determined the structure of zebrafish STRA6 in complex with CaM to 3.9 Å resolution. The protein is assembled as an intricate dimer with a topology that includes 18 TM helices (nine per protomer) and two long horizontal intramembrane (IM) helices interacting at the dimer core. Each STRA6 protomer comprises an N-terminal domain (NTD) of the first five TM helices, connected by a linker containing the first CaM-binding peptide to a central domain at the dimer interface that includes TMs 6 to 9 and the IM helices, and a cytoplasmic C-terminal segment that interacts with CaM through two additional helices. Each protomer is compactly associated with one molecule of CaM, adopting an unconventional arrangement in which it is bound to three helical regions of STRA6. We characterized the STRA6-CaM interaction biophysically by isothermal titration calorimetry, showing that the affinity of CaM for one STRA6 peptide alone is subnanomolar, and structurally by x-ray crystallography. We also demonstrated that the STRA6-CaM association is physiological by performing immunoprecipitation experiments on native zebrafish tissue. Both the extra- and intracellular surfaces of the NTD feature conserved polar pockets. The outer NTD pocket spans half the bilayer. The central domain of STRA6 defines a large ~20,000 Å3 cleft on the extracellular side, which encompasses the space between previously characterized binding sites for RBP, ~25 Å above the membrane surface, and the IM helices located down at the mid-bilayer level. This outer cleft is hydrophobic, contains two ordered putative cholesterols, and is exposed to the membrane through two symmetry-related lateral windows defined by TMs 8 and 9 and the IM helices. CONCLUSIONS The structure of STRA6 suggests a mechanism for retinol release from RBP into the hydrophobic environment of the outer cleft and direct diffusion into the membrane through the lateral window. Our work also sets the basis for future experiments aimed at investigating how the system is regulated, whether STRA6 also has a role in signaling, and the functional relevance of its association with CaM. The structure of STRA6 in complex with CaM. The STRA6 dimer, drawn as a ribbon representation with one protomer in dark red and the other in black, is associated with two molecules of calmodulin, drawn in gray and gold. The internal volume of the outer cleft is represented as a semitransparent blue surface. Calcium ions are represented as green spheres. Vitamin A homeostasis is critical to normal cellular function. Retinol-binding protein (RBP) is the sole specific carrier in the bloodstream for hydrophobic retinol, the main form in which vitamin A is transported. The integral membrane receptor STRA6 mediates cellular uptake of vitamin A by recognizing RBP-retinol to trigger release and internalization of retinol. We present the structure of zebrafish STRA6 determined to 3.9-angstrom resolution by single-particle cryo-electron microscopy. STRA6 has one intramembrane and nine transmembrane helices in an intricate dimeric assembly. Unexpectedly, calmodulin is bound tightly to STRA6 in a noncanonical arrangement. Residues involved with RBP binding map to an archlike structure that covers a deep lipophilic cleft. This cleft is open to the membrane, suggesting a possible mode for internalization of retinol through direct diffusion into the lipid bilayer.

Serum retinol-binding protein 4 (RBP4) and retinol in a cohort of borderline obese women with and without gestational diabetes.

OBJECTIVES To evaluate whether serum RBP4 correlates with gestational diabetes mellitus (GDM) in a cohort of borderline obese (BMI>30) pregnant women. DESIGN AND METHODS Serum RBP4 and retinol were measured in pregnant women with (n=12) and without (n=10) GDM. RESULTS RBP4, retinol and RBP4:retinol molar ratio were not different between the groups and were not associated with markers of insulin resistance. CONCLUSIONS GDM is not associated with RBP4 or retinol among borderline obese pregnant women.

β-Apo-10'-carotenoids Modulate Placental Microsomal Triglyceride Transfer Protein Expression and Function to Optimize Transport of Intact β-Carotene to the Embryo.

β-Carotene is an important source of vitamin A for the mammalian embryo, which depends on its adequate supply to achieve proper organogenesis. In mammalian tissues, β-carotene 15,15′-oxygenase (BCO1) converts β-carotene to retinaldehyde, which is then oxidized to retinoic acid, the biologically active form of vitamin A that acts as a transcription factor ligand to regulate gene expression. β-Carotene can also be cleaved by β-carotene 9′,10′-oxygenase (BCO2) to form β-apo-10′-carotenal, a precursor of retinoic acid and a transcriptional regulator per se. The mammalian embryo obtains β-carotene from the maternal circulation. However, the molecular mechanisms that enable its transfer across the maternal-fetal barrier are not understood. Given that β-carotene is transported in the adult bloodstream by lipoproteins and that the placenta acquires, assembles, and secretes lipoproteins, we hypothesized that the aforementioned process requires placental lipoprotein biosynthesis. Here we show that β-carotene availability regulates transcription and activity of placental microsomal triglyceride transfer protein as well as expression of placental apolipoprotein B, two key players in lipoprotein biosynthesis. We also show that β-apo-10′-carotenal mediates the transcriptional regulation of microsomal triglyceride transfer protein via hepatic nuclear factor 4α and chicken ovalbumin upstream promoter transcription factor I/II. Our data provide the first in vivo evidence of the transcriptional regulatory activity of β-apocarotenoids and identify microsomal triglyceride transfer protein and its transcription factors as the targets of their action. This study demonstrates that β-carotene induces a feed-forward mechanism in the placenta to enhance the assimilation of β-carotene for proper embryogenesis.

Retinol as a cofactor for PKCδ-mediated impairment of insulin sensitivity in a mouse model of diet-induced obesity

We previously defined that the mitochondria‐localized PKCδ signaling complex stimulates the conversion of pyruvate to acetyl‐coenzyme A by the pyruvate dehydrogenase complex. We demonstrated in vitro and ex vivo that retinol supplementation enhances ATP synthesis in the presence of the PKCδ signalosome. Here, we tested in vivo if a persistent oversupply of retinol would further impair glucose metabolism in a mouse model of diet‐induced insulin resistance. We crossed mice over‐expressing human retinol‐binding protein (hRBP) under the muscle creatine kinase (MCK) promoter (MCKhRBP) with the PKCδ–/– strain to generate mice with a different status of the PKCδ signalosome and retinoid levels. Mice with a functional PKCδ signalosome and elevated retinoid levels (PKCδ+/+hRBP) developed the most advanced stage of insulin resistance. In contrast, elevation of retinoid levels in mice with inactive PKCδ did not affect remarkably their metabolism, resulting in phenotypic similarity between PKCδ–/– hRBP and PKCδ–/– mice. Therefore, in addition to the well‐defined role of PKCδ in the etiology of metabolic syndrome, we present a novel PKCδ signaling pathway that requires retinol as a metabolic cofactor and is involved in the regulation of fuel utilization in mitochondria. The distinct role in whole‐body energy homeostasis establishes the PKCδ signalosome as a promising target for therapeutic intervention in metabolic disorders.—Shabrova, E., Hoyos, B., Vinogradov, V., Kim, Y.‐K., Wassef, L., Leitges, M., Quadro, L., Hammerling, U., Retinol as a cofactor for PKCδ‐mediated impairment of insulin sensitivity in a mouse model of diet‐induced obesity. FASEB J. 30, 1339–1355 (2016). www.fasebj.org

Low-Density Lipoprotein Receptor Contributes to β-Carotene Uptake in the Maternal Liver

Vitamin A regulates many essential mammalian biological processes, including embryonic development. β-carotene is the main source of vitamin A in the human diet. Once ingested, it is packaged into lipoproteins, predominantly low-density lipoproteins (LDL), and transported to different sites within the body, including the liver and developing tissues, where it can either be stored or metabolized to retinoids (vitamin A and its derivatives). The molecular mechanisms of β-carotene uptake by the liver or developing tissues remain elusive. Here, we investigated the role of the LDL receptor (LDLr) in β-carotene uptake by maternal liver, placenta and embryo. We administered a single dose of β-carotene to Ldlr+/− and Ldlr−/− pregnant mice via intraperitoneal injection at mid-gestation and monitored the changes in β-carotene content among maternal lipoproteins and the liver, as well as the accumulation of β-carotene in the placental–fetal unit. We showed an abnormal β-carotene distribution among serum lipoproteins and reduced hepatic β-carotene uptake in Ldlr−/− dams. These data strongly imply that LDLr significantly contributes to β-carotene uptake in the adult mouse liver. In contrast, LDLr does not seem to mediate acquisition of β-carotene by the placental–fetal unit.