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Investigative Ophthalmology & Visual... Jan 2017Two-photon excited fluorescence (TPEF) imaging has potential as a functional tool for tracking visual pigment regeneration in the living eye. Previous studies have shown...
PURPOSE
Two-photon excited fluorescence (TPEF) imaging has potential as a functional tool for tracking visual pigment regeneration in the living eye. Previous studies have shown that all-trans-retinol is likely the chief source of time-varying TPEF from photoreceptors. Endogenous TPEF from retinol could provide the specificity desired for tracking the visual cycle. However, in vivo characterization of native retinol kinetics is complicated by visual stimulation from the imaging beam. We have developed an imaging scheme for overcoming these challenges and monitored the formation and clearance of retinol.
METHODS
Three macaques were imaged by using an in vivo two-photon ophthalmoscope. Endogenous TPEF was excited at 730 nm and recorded through the eye's pupil for more than 90 seconds. Two-photon excited fluorescence increased with onset of light and plateaued within 40 seconds, at which point, brief incremental stimuli were delivered at 561 nm. The responses of rods to stimulation were analyzed by using first-order kinetics.
RESULTS
Two-photon excited fluorescence resulting from retinol production corresponded to the fraction of rhodopsin bleached. The photosensitivity of rhodopsin was estimated to be 6.88 ± 5.50 log scotopic troland. The rate of retinol clearance depended on intensity of incremental stimulation. Clearance was faster for stronger stimuli and time constants ranged from 50 to 300 seconds.
CONCLUSIONS
This study demonstrates a method for rapidly measuring the rate of clearance of retinol in vivo. Moreover, TPEF generated due to retinol can be used as a measure of rhodopsin depletion, similar to densitometry. This enhances the utility of two-photon ophthalmoscopy as a technique for evaluating the visual cycle in the living eye.
Topics: Animals; Dark Adaptation; Female; Macaca fascicularis; Male; Models, Animal; Ophthalmoscopy; Optical Imaging; Retinal Pigments; Retinal Rod Photoreceptor Cells; Retinol-Binding Proteins; Rhodopsin; Vitamin A
PubMed: 28129424
DOI: 10.1167/iovs.16-20061 -
The Journal of Biological Chemistry Nov 2004Retinoids carry out essential functions in vertebrate development and vision. Many of the retinoid processing enzymes remain to be identified at the molecular level. To...
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.
Topics: Amino Acid Sequence; Animals; Chromatography, High Pressure Liquid; Cloning, Molecular; Enzymes; Solanum lycopersicum; Macaca fascicularis; Mass Spectrometry; Mice; Microsomes; Molecular Sequence Data; Sequence Alignment; Time Factors; Vitamin A
PubMed: 15358783
DOI: 10.1074/jbc.M409130200 -
Biomolecules Dec 2019The concentration of all--retinoic acid, the bioactive derivative of vitamin A, is critically important for the optimal performance of numerous physiological processes.... (Review)
Review
The concentration of all--retinoic acid, the bioactive derivative of vitamin A, is critically important for the optimal performance of numerous physiological processes. Either too little or too much of retinoic acid in developing or adult tissues is equally harmful. All--retinoic acid is produced by the irreversible oxidation of all--retinaldehyde. Thus, the concentration of retinaldehyde as the immediate precursor of retinoic acid has to be tightly controlled. However, the enzymes that produce all--retinaldehyde for retinoic acid biosynthesis and the mechanisms responsible for the control of retinaldehyde levels have not yet been fully defined. The goal of this review is to summarize the current state of knowledge regarding the identities of physiologically relevant retinol dehydrogenases, their enzymatic properties, and tissue distribution, and to discuss potential mechanisms for the regulation of the flux from retinol to retinaldehyde.
Topics: Animals; Biosynthetic Pathways; Humans; Retinaldehyde; Tretinoin
PubMed: 31861321
DOI: 10.3390/biom10010005 -
The Journal of Biological Chemistry Nov 2017Interphotoreceptor retinoid-binding protein (IRBP) is a specialized lipophilic carrier that binds the all- and 11- isomers of retinal and retinol, and this facilitates...
Interphotoreceptor retinoid-binding protein (IRBP) is a specialized lipophilic carrier that binds the all- and 11- isomers of retinal and retinol, and this facilitates their transport between photoreceptors and cells in the retina. One of these retinoids, all-retinal, is released in the rod outer segment by photoactivated rhodopsin after light excitation. Following its release, all-retinal is reduced by the retinol dehydrogenase RDH8 to all-retinol in an NADPH-dependent reaction. However, all-retinal can also react with outer segment components, sometimes forming lipofuscin precursors, which after conversion to lipofuscin accumulate in the lysosomes of the retinal pigment epithelium and display cytotoxic effects. Here, we have imaged the fluorescence of all-retinol, all-retinal, and lipofuscin precursors in real time in single isolated mouse rod photoreceptors. We found that IRBP removes all-retinol from individual rod photoreceptors in a concentration-dependent manner. The rate constant for retinol removal increased linearly with IRBP concentration with a slope of 0.012 min μm IRBP also removed all-retinal, but with much less efficacy, indicating that the reduction of retinal to retinol promotes faster clearance of the photoisomerized rhodopsin chromophore. The presence of physiological IRBP concentrations in the extracellular medium resulted in lower levels of all-retinal and retinol in rod outer segments following light exposure. It also prevented light-induced lipofuscin precursor formation, but it did not remove precursors that were already present. These findings reveal an important and previously unappreciated role of IRBP in protecting the photoreceptor cells against the cytotoxic effects of accumulated all-retinal.
Topics: Animals; Cattle; Eye Proteins; Light; Lipofuscin; Mice; Mice, Knockout; Retinaldehyde; Retinol-Binding Proteins; Rod Cell Outer Segment; Vitamin A
PubMed: 28972139
DOI: 10.1074/jbc.M117.795187 -
Bioscience, Biotechnology, and... Oct 2008We assessed the effects of ATRA and retinol on melanogenesis in murine B16 melanoma cells. In the present study, ATRA and retinol inhibited melanin synthesis in melanoma...
We assessed the effects of ATRA and retinol on melanogenesis in murine B16 melanoma cells. In the present study, ATRA and retinol inhibited melanin synthesis in melanoma cells stimulated by alpha-melanocyte stimulating hormone (alpha-MSH) or 3-isobutyl-1-methylxanthine (IBMX). To elucidate the target points of ATRA and retinol on melanogenesis, we performed western blotting for melanogenic proteins, such as tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. ATRA inhibited the expression of tyrosinase and TRP-1, and retinol inhibited the expression of tyrosinase, in a dose-dependent manner. Neither ATRA nor retinol inhibited TRP-2 expression. There were no differences in melanogenic protein expression between the two stimulants tested, alpha-MSH and IBMX. Therefore, the depigmenting effect of ATRA and retinol might be due to inhibition of the signaling pathway between cAMP and tyrosinase transcription bound to tyrosinase expression. These results indicate that ATRA and retinol are candidate anti-melanogenic agents that they might be effective in hyperpigmentation disorders.
Topics: Animals; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Melanins; Melanoma; Mice; Monophenol Monooxygenase; Pigmentation; Tretinoin; Vitamin A
PubMed: 18838813
DOI: 10.1271/bbb.80279 -
Nutrients Mar 2022Vitamin A (VA), all-trans-retinol (ROL), and its analogs are collectively called retinoids. Acting through the retinoic acid receptors RARα, RARβ, and RARγ,... (Review)
Review
Vitamin A (VA), all-trans-retinol (ROL), and its analogs are collectively called retinoids. Acting through the retinoic acid receptors RARα, RARβ, and RARγ, all-trans-retinoic acid, an active metabolite of VA, is a potent regulator of numerous biological pathways, including embryonic and somatic cellular differentiation, immune functions, and energy metabolism. The liver is the primary organ for retinoid storage and metabolism in humans. For reasons that remain incompletely understood, a body of evidence shows that reductions in liver retinoids, aberrant retinoid metabolism, and reductions in RAR signaling are implicated in numerous diseases of the liver, including hepatocellular carcinoma, non-alcohol-associated fatty liver diseases, and alcohol-associated liver diseases. Conversely, restoration of retinoid signaling, pharmacological treatments with natural and synthetic retinoids, and newer agonists for specific RARs show promising benefits for treatment of a number of these liver diseases. Here we provide a comprehensive review of the literature demonstrating a role for retinoids in limiting the pathogenesis of these diseases and in the treatment of liver diseases.
Topics: Humans; Liver Diseases; Receptors, Retinoic Acid; Retinoids; Tretinoin; Vitamin A
PubMed: 35406069
DOI: 10.3390/nu14071456 -
Nutrients Dec 2021Fat-soluble vitamin deficiency remains a challenge in cystic fibrosis (CF), chronic pancreatitis, and biliary atresia. Liposomes and cyclodextrins can enhance their... (Randomized Controlled Trial)
Randomized Controlled Trial
Fat-soluble vitamin deficiency remains a challenge in cystic fibrosis (CF), chronic pancreatitis, and biliary atresia. Liposomes and cyclodextrins can enhance their bioavailability, thus this multi-center randomized placebo-controlled trial compared three-month supplementation of fat-soluble vitamins in the form of liposomes or cyclodextrins to medium-chain triglycerides (MCT) in pancreatic-insufficient CF patients. The daily doses were as follows: 2000 IU of retinyl palmitate, 4000 IU of vitamin D3, 200 IU of RRR-α-tocopherol, and 200 µg of vitamin K2 as menaquinone-7, with vitamin E given in soybean oil instead of liposomes. All participants received 4 mg of β-carotene and 1.07 mg of vitamin K1 to ensure compliance with the guidelines. The primary outcome was the change from the baseline of all-trans-retinol and 25-hydroxyvitamin D3 concentrations and the percentage of undercarboxylated osteocalcin. Out of 75 randomized patients ( = 28 liposomes, = 22 cyclodextrins, and = 25 MCT), 67 completed the trial (89%; = 26 liposomes, = 18 cyclodextrins, and = 23 MCT) and had a median age of 22 years (IQR 19-28), body mass index of 20.6 kg/m [18.4-22.0], and forced expiratory volume in 1 s of 65% (44-84%). The liposomal formulation of vitamin A was associated with the improved evolution of serum all-trans-retinol compared to the control (median +1.7 ng/mL (IQR -44.3-86.1) vs. -38.8 ng/mL (-71.2-6.8), = 0.028). Cyclodextrins enhanced the bioavailability of vitamin D3 (+9.0 ng/mL (1.0-17.0) vs. +3.0 ng/mL (-4.0-7.0), = 0.012) and vitamin E (+4.34 µg/mL (0.33-6.52) vs. -0.34 µg/mL (-1.71-2.15), = 0.010). Liposomes may augment the bioavailability of vitamin A and cyclodextrins may strengthen the supplementation of vitamins D3 and E relative to MCT in pancreatic-insufficient CF but further studies are required to assess liposomal vitamin E (German Clinical Trial Register number DRKS00014295, funded from EU and Norsa Pharma).
Topics: Adolescent; Adult; Calcifediol; Cholecalciferol; Cyclodextrins; Cystic Fibrosis; Dietary Supplements; Exocrine Pancreatic Insufficiency; Female; Humans; Liposomes; Male; Treatment Outcome; Triglycerides; Vitamin A; Vitamin D; Vitamin E; Vitamin K 2; Vitamins; Young Adult; beta Carotene
PubMed: 34960106
DOI: 10.3390/nu13124554 -
The Journal of Nutrition Feb 2010Recent investigations have demonstrated that elevated serum retinol-binding protein 4 (RBP4) secreted from adipose tissue plays a role in the development of systemic...
Recent investigations have demonstrated that elevated serum retinol-binding protein 4 (RBP4) secreted from adipose tissue plays a role in the development of systemic insulin resistance, and lowering RBP4 improves insulin sensitivity. These observations provide a rationale for the development of new antidiabetic agents aimed at reducing serum RBP4 concentrations. In this study, we sought to determine whether retinoic acid (RA) administration decreases serum RBP4 and suppresses insulin resistance in diabetic ob/ob mice. All-trans RA [100 mug/(moused) in corn oil] was administered by stomach intubation to a group of ob/ob mice, with the control group receiving the vehicle for 16 d. Body weight and food intake were monitored. Glucose and insulin tolerance tests were performed. We quantified serum RBP4 and retinol by Western blotting and HPLC, respectively. RA treatment reduced body weight (P < 0.05), basal serum glucose (P < 0.001), serum retinol (P < 0.01), and RBP4 (P < 0.05). It improved insulin sensitivity and decreased the retinol:RBP4 ratio (P < 0.05). These studies suggest that RA is an effective antidiabetic agent that could be considered in the treatment of type 2 diabetes.
Topics: Animals; Blood Glucose; Blotting, Western; Body Weight; Chromatography, High Pressure Liquid; Diabetes Mellitus, Experimental; Glucose Tolerance Test; Hypoglycemic Agents; Insulin Resistance; Mice; Mice, Obese; Retinol-Binding Proteins, Plasma; Tretinoin; Vitamin A
PubMed: 20032483
DOI: 10.3945/jn.109.115147 -
Nutrients Nov 2016The visual system produces visual chromophore, 11--retinal from dietary vitamin A, all--retinol making this vitamin essential for retinal health and function. These... (Review)
Review
The visual system produces visual chromophore, 11--retinal from dietary vitamin A, all--retinol making this vitamin essential for retinal health and function. These metabolic events are mediated by a sequential biochemical process called the visual cycle. Retinol dehydrogenases (RDHs) are responsible for two reactions in the visual cycle performed in retinal pigmented epithelial (RPE) cells, photoreceptor cells and Müller cells in the retina. RDHs in the RPE function as 11--RDHs, which oxidize 11--retinol to 11--retinal in vivo. RDHs in rod photoreceptor cells in the retina work as all--RDHs, which reduce all--retinal to all--retinol. Dysfunction of RDHs can cause inherited retinal diseases in humans. To facilitate further understanding of human diseases, mouse models of RDHs-related diseases have been carefully examined and have revealed the physiological contribution of specific RDHs to visual cycle function and overall retinal health. Herein we describe the function of RDHs in the RPE and the retina, particularly in rod photoreceptor cells, their regulatory properties for retinoid homeostasis and future therapeutic strategy for treatment of retinal diseases.
Topics: Alcohol Oxidoreductases; Animals; Ependymoglial Cells; Genetic Predisposition to Disease; Humans; Mutation; Oxidation-Reduction; Phenotype; Retinal Diseases; Retinal Pigment Epithelium; Retinal Rod Photoreceptor Cells; Retinaldehyde; Vision, Ocular; Vitamin A
PubMed: 27879662
DOI: 10.3390/nu8110746 -
The EMBO Journal Sep 1998The potential storage and delivery function of cartilage oligomeric matrix protein (COMP) for cell signaling molecules was explored by binding hydrophobic compounds to...
The potential storage and delivery function of cartilage oligomeric matrix protein (COMP) for cell signaling molecules was explored by binding hydrophobic compounds to the recombinant five-stranded coiled-coil domain of COMP. Complex formation with benzene, cyclohexane, vitamin D3 and elaidic acid was demonstrated through increases in denaturation temperatures of 2-10 degreesC. For all-trans retinol and all-trans retinoic acid, an equilibrium dissociation constant KD = 0.6 microM was evaluated by fluorescence titration. Binding of benzene and all-trans retinol into the hydrophobic axial pore of the COMP coiled-coil domain was proven by the X-ray crystal structures of the corresponding complexes at 0.25 and 0.27 nm resolution, respectively. Benzene binds with its plane perpendicular to the pore axis. The binding site is between the two internal rings formed by Leu37 and Thr40 pointing into the pore of the COMP coiled-coil domain. The retinol beta-ionone ring is positioned in a hydrophobic environment near Thr40, and the 1.1 nm long isoprene tail follows a completely hydrophobic region of the pore. Its terminal hydroxyl group complexes with a ring of the five side chains of Gln54. A mutant in which Gln54 is replaced by Ile binds all-trans retinol with affinity similar to the wild-type, demonstrating that hydrophobic interactions are predominant.
Topics: Amino Acid Sequence; Animals; Benzene; Binding Sites; Cholecalciferol; Crystallography, X-Ray; Cyclohexanes; Extracellular Matrix Proteins; Glycoproteins; Matrilin Proteins; Models, Molecular; Molecular Sequence Data; Oleic Acid; Oleic Acids; Protein Conformation; Protein Denaturation; Rats; Recombinant Fusion Proteins; Spectrometry, Fluorescence; Tretinoin
PubMed: 9736606
DOI: 10.1093/emboj/17.18.5265