quantitative-analysis-dietary-vitamin-metabolites-murine-ocular-non

Quantitative Analysis of Dietary Vitamin A Metabolites in Murine Ocular and Non-Ocular Tissues Using High-Performance Liquid Chromatography

G protein-coupled receptors (GPCRs) are a superfamily of transmembrane proteins that initiate signaling cascades through activation of its G protein upon ...

University of Minnesota

Vitamins are essential compounds obtained through diet that are necessary for normal development and function in an organism. One of the most important vitamins for human physiology is vitamin A, a group of retinoid compounds and carotenoids, which generally function as a mediator for cell growth, differentiation, immunity, and embryonic development, as well as serving as a key component in the phototransduction cycle in the vertebrate retina. For humans, vitamin A is obtained through the diet, where provitamin A carotenoids such as β-carotene from plants or preformed vitamin A such as retinyl esters from animal sources are absorbed into the body via the small intestine and converted into all- retinol within the intestinal enterocytes. Specifically, once absorbed, carotenoids are cleaved by carotenoid cleavage oxygenases (CCOs), such as Beta-carotene 15,15'-monooxygenase (BCO1), to produce all- retinal that subsequently gets converted into all- retinol. CRBP2 bound retinol is then converted into retinyl esters (REs) by the enzyme lecithin retinol acyltransferase (LRAT) in the endoplasmic reticulum, which is then packaged into chylomicrons and sent into the bloodstream for storage in hepatic stellate cells in the liver or for functional use in peripheral tissues such as the retina. All-trans retinol also travels through the bloodstream bound to retinol binding protein 4 (RBP4), where it enters cells with the assistance of the transmembrane transporters, stimulated by retinoic acid 6 (STRA6) in peripheral tissues or retinol binding protein 4 receptor 2 (RBPR2) in systemic tissues (e.g., in the retina and the liver, respectively). Much is known about the intake, metabolism, storage, and function of vitamin A compounds, especially with regard to its impact on eye development and visual function in the retinoid cycle. However, there is much to learn about the role of vitamin A as a transcription factor in development and cell growth, as well as how peripheral cells signal hepatocytes to secrete all- retinol into the blood for peripheral cell use. This article aims to review literature regarding the major known pathways of vitamin A intake from dietary sources into hepatocytes, vitamin A excretion by hepatocytes, as well as vitamin A usage within the retinoid cycle in the RPE and retina to provide insight on future directions of novel membrane transporters for vitamin A in retinal cell physiology and visual function.

Vitamin A Transporters in Visual Function: A Mini Review on Membrane Receptors for Dietary Vitamin A Uptake, Storage, and Transport to the Eye.

Rods and cones are photoreceptor neurons in the retina that are required for visual sensation in vertebrates, wherein the perception of vision is initiated when these neurons respond to photons in the light stimuli. The photoreceptor cell is structurally studied as outer segments (OS) and inner segments (IS) where proper protein sorting, localization, and compartmentalization are critical for phototransduction, visual function, and survival. In human retinal diseases, improper protein transport to the OS or mislocalization of proteins to the IS and other cellular compartments could lead to impaired visual responses and photoreceptor cell degeneration that ultimately cause loss of visual function.

The role of motor proteins in photoreceptor protein transport and visual function.

The systemic transport of dietary vitamin A/all- retinol bound to RBP4 into peripheral tissues for storage is an essential physiological process that continuously provides visual chromophore precursors to the retina under fasting conditions. This mechanism is critical for phototransduction, photoreceptor cell maintenance and survival, and in the support of visual function. While the membrane receptor STRA6 facilitates the blood transport of lipophilic vitamin A into the eye, it is not expressed in most peripheral organs, which are proposed to express a second membrane receptor for the uptake of vitamin A from circulating RBP4. The discovery of a novel vitamin A receptor, RBPR2, which is expressed in the liver and intestine, but not in the eye, alluded to this long-sort non-ocular membrane receptor for systemic RBP4-ROL uptake and transport. We have previously shown in zebrafish that the retinol-binding protein receptor 2 (Rbpr2) plays an important role in the transport of yolk vitamin A to the eye. Mutant  zebrafish lines manifested in decreased ocular retinoid concentrations and retinal phenotypes. To investigate a physiological role for the second vitamin A receptor, RBPR2, in mammals and to analyze the metabolic basis of systemic vitamin A transport for retinoid homeostasis, we established a whole-body  knockout mouse () model. These mice were viable on both vitamin A-sufficient and -deficient diets.  mice that were fed a vitamin A-sufficient diet displayed lower ocular retinoid levels, decreased opsins, and manifested in decrease visual function, as measured by electroretinography. Interestingly, when  mice were fed a vitamin A-deficient diet, they additionally showed shorter photoreceptor outer segment phenotypes, altogether manifesting in a significant loss of visual function. Thus, under conditions replicating vitamin A sufficiency and deficiency, our analyses revealed that RBPR2-mediated systemic vitamin A transport is a regulated process that is important for vitamin A delivery to the eye when RBP4-bound ROL is the only transport pathway in the fasting condition or under vitamin A deficiency conditions.

Mice Lacking the Systemic Vitamin A Receptor RBPR2 Show Decreased Ocular Retinoids and Loss of Visual Function.

The distribution of dietary vitamin A/all- retinol/ROL throughout the body is critical for maintaining retinoid function in peripheral tissues and for retinoid delivery to the eye in the support of visual function. In the circulation, all--retinol bound to the RBP4 protein is transported and sequestered into target tissues for long-term storage. Two membrane receptors that facilitate all- retinol uptake from RBP4 have been proposed. While it is well established that the membrane receptor, STRA6, binds to circulatory RBP4 for ROL transport into the eye, the second vitamin A receptor, RBPR2, which is expressed in non-ocular tissues, is less characterized. Based on the structural homology between these two RBP4 receptors, published literature, and from our recent work in   deficient mice, we hypothesized that RBPR2 might also have high-binding affinity for RBP4 and this mechanism facilitates ROL transport. Herein, we aimed to elucidate the membrane topology and putative RBP4 binding residues on RBPR2 to understand its physiological function for retinoid homeostasis. Using  analysis and site-directed mutagenesis, we identified a potential RBP4 binding domain on RBPR2. We employed an  cell-based system and confirmed that mutations of these residues on RBPR2 affected its binding to exogenous RBP4 and subsequently vitamin A uptake. Using Surface Plasmon Resonance assays, we analyzed both the binding affinities and kinetic parameters of wild-type RBPR2 and individual mutants affecting the RBPR2-RBP4 binding domain with its physiological ligand RBP4. These studies not only revealed a putative RBP4 binding domain on RBPR2 but also provided new structural, biochemical, and critical information on its proposed role in RBP4 binding for ROL transport and retinoid homeostasis.

Mapping of the extracellular RBP4 ligand binding domain on the RBPR2 receptor for Vitamin A transport.

Rods and cones are photoreceptor neurons in the retina that are required for visual sensation in vertebrates, where proper protein localization and compartmentalization are critical for phototransduction and visual function. In human retinal diseases, improper protein transport to the outer segment (OS) or mislocalization of proteins to the inner segment (IS) could lead to impaired visual responses and photoreceptor cell degeneration, causing a loss of visual function. We showed involvement of an unconventional motor protein, MYO1C, in the proper localization of rhodopsin to the OS, where loss of MYO1C in a mammalian model caused mislocalization of rhodopsin to IS and cell bodies, leading to progressively severe retinal phenotypes. In this study, using modeling and docking analysis, we aimed to identify the protein-protein interaction sites between MYO1C and Rhodopsin to establish a hypothesis that a physical interaction between these proteins is necessary for the proper trafficking of rhodopsin and visual function.

In Silico Prediction of MYO1C-Rhodopsin Interactions and Its Significance in Protein Localization and Visual Function.

In this review, we outline our current understanding of the mechanisms involved in the absorption, storage, and transport of dietary vitamin A to the eye, and the trafficking of rhodopsin protein to the photoreceptor outer segments, which encompasses the logistical backbone required for photoreceptor cell function. Two key mechanisms of this process are emphasized in this manuscript: ocular and systemic vitamin A membrane transporters, and rhodopsin transporters. Understanding the complementary mechanisms responsible for the generation and proper transport of the retinylidene protein to the photoreceptor outer segment will eventually shed light on the importance of genes encoded by these proteins, and their relationship on normal visual function and in the pathophysiology of retinal degenerative diseases.

The Logistical Backbone of Photoreceptor Cell Function: Complementary Mechanisms of Dietary Vitamin A Receptors and Rhodopsin Transporters.

The distribution of dietary vitamin A/all- retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and for generating visual pigments for photoreceptor cell function. ROL circulates in the blood bound to the retinol binding protein 4 (RBP4) as RBP4-ROL. Two membrane receptors, RBPR2 in the liver and STRA6 in the eye are proposed to bind circulatory RBP4 and this mechanism is critical for internalizing ROL into cells. Here, we present a longitudinal investigation towards the importance of RBPR2 and influence of the diet on systemic retinoid homeostasis for visual function. Age matched -KO (  ) and wild-type (WT) mice were fed either a vitamin A sufficient (VAS) or a vitamin A deficient (VAD) diet. At 3- and 6-months, we performed retinoid quantification of ocular and non-ocular tissues using HPLC analysis and complemented the data with visual physiology, rhodopsin quantification by spectrophotometry, and biochemical analysis. At 3-months and compared to WT mice,   mice fed either vitamin A diets displayed lower scotopic and photopic electroretinogram (ERG) responses, which correlated with HPLC analysis that revealed   mice had significantly lower hepatic and ocular retinoid content. Interestingly, with the exception of the liver, long-term feeding of   mice with a VAS diet promoted all- retinol accumulation in most peripheral tissues. However, even under VAS dietary conditions significant amounts of unliganded opsins in rods, together with decreased visual responses were evident in aged mice lacking RBPR2, when compared to WT mice. Together, our analyses characterize the molecular events underlying nutritional blindness in a novel mouse model and indicate that loss of the liver specific RBP4-ROL receptor, RBPR2, influences systemic retinoid homeostasis and rhodopsin synthesis, which causes profound visual function defects under severe vitamin A deficiency conditions.

Article	Year
Vitamin A Transporters in Visual Function: A Mini Review on Membrane Receptors for Dietary Vitamin A Uptake, Storage, and Transport to the Eye. Nutrients\| PubMed ID: 34836244	2021
The role of motor proteins in photoreceptor protein transport and visual function. Ophthalmic genetics\| PubMed ID: 35470760	2022
Mice Lacking the Systemic Vitamin A Receptor RBPR2 Show Decreased Ocular Retinoids and Loss of Visual Function. Nutrients\| PubMed ID: 35745101	2022
Mapping of the extracellular RBP4 ligand binding domain on the RBPR2 receptor for Vitamin A transport. Frontiers in cell and developmental biology\| PubMed ID: 36910150	2023
In Silico Prediction of MYO1C-Rhodopsin Interactions and Its Significance in Protein Localization and Visual Function. Advances in experimental medicine and biology\| PubMed ID: 37440078	2023
The Logistical Backbone of Photoreceptor Cell Function: Complementary Mechanisms of Dietary Vitamin A Receptors and Rhodopsin Transporters. International journal of molecular sciences\| PubMed ID: 38673863	2024
Loss of the systemic vitamin A transporter RBPR2 affects the quantitative balance between chromophore and opsins in visual pigment synthesis. bioRxiv : the preprint server for biology\| PubMed ID: 39026765	2024

Matthias Leung

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