The Drosophila class B scavenger receptor NinaD-I is a cell surface receptor mediating carotenoid transport for visual chromophore synthesis

The blind Drosophila mutant ninaD lacks the visual chromophore. Genetic evidence that the molecular basis is a defect in carotenoid uptake which causes vitamin A deficiency exists. The ninaD gene encodes a scavenger receptor that is significantly homologous in sequence with the mammalian scavenger receptors SR-BI (scavenger receptor class B type I) and CD36 (cluster determinant 36), yet NinaD has not been characterized in functional detail. Therefore, we established a Drosophila S2 cell culture system for biochemically characterizing the ninaD gene products. We show that the two splice variant isoforms encoded by ninaD exhibit different subcellular localizations. NinaD-I, the long protein variant, is localized at the plasma membrane, whereas the short variant, NinaD-II, is localized at intracellular membranes. Only NinaD-I could mediate the cellular uptake of carotenoids from micelles in this cell culture system. Carotenoid uptake was concentration-dependent and saturable. By in vivo analyses of different mutant and transgenic fly strains, we provide evidence of an essential role of NinaD-I in the absorption of dietary carotenoids to support visual chromophore synthesis. Moreover, our analyses suggest a role of NinaD-I in tocopherol metabolism. Even though Drosophila is a sterol auxotroph, we found no evidence of a contribution of NinaD-I to the uptake of these compounds. Together, our study establishes an evolutionarily conserved connection between class B scavenger receptors and the numerous functions of fat soluble vitamins in animal physiology.     

Mechanisms involved in the intestinal absorption of dietary vitamin A and provitamin A carotenoids

Vitamin A is an essential nutrient for humans and is converted to the visual chromophore, 11-cis-retinal, and to the hormone, retinoic acid. Vitamin A in animal-derived foods is found as long chain acyl esters of retinol and these are digested to free fatty acids and retinol before uptake by the intestinal mucosal cell. The retinol is then reesterified to retinyl esters for incorporation into chlylomicrons and absorbed via the lymphatics or effluxed into the portal circulation facilitated by the lipid transporter, ABCA1. Provitamin A carotenoids such as β-carotene are found in plant-derived foods. These and other carotenoids are transported into the mucosal cell by scavenger receptor class B type I (SR-BI). Provitamin A carotenoids are partly converted to retinol by oxygenase and reductase enzymes and the retinol so produced is available for absorption via the two pathways described above. The efficiency of vitamin A and carotenoid intestinal absorption is determined by the regulation of a number of proteins involved in the process. Polymorphisms in genes for these proteins lead to individual variability in the metabolism and transport of vitamin A and carotenoids. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.     

Class B scavenger receptor-mediated intestinal absorption of dietary beta-carotene and cholesterol

There is now a general consensus that the intestinal absorption of water-insoluble, dietary lipids is protein-mediated, but the assignment of protein(s) to this function is still a matter of debate. To address this issue, we measured beta-carotene and cholesterol absorption in wild-type and SR-BI knockout mice and the uptake of these lipids in vitro using brush border membrane (BBM) vesicles. From the comparison of the in vivo and in vitro results we conclude that both BBM-resident class B scavenger receptors, SR-BI and CD36, can facilitate the absorption of beta-carotene and cholesterol. SR-BI is essential for beta-carotene absorption, at least in mice on a high fat diet. This is due to the fact that the absorption of beta-carotene is restricted to the duodenum and SR-BI is the predominant receptor in the mouse duodenum. In contrast, SR-BI may be involved but is not essential for cholesterol absorption in the small intestine. The question of whether SR-BI contributes to cholesterol absorption in vivo is still unresolved. Transfection of COS-7 cells with SR-BI or CD36 confers on these cells lipid uptake properties closely resembling those of enterocytes and BBM vesicles. Both scavenger receptors facilitate the uptake of dietary lipids such as beta-carotene, free and esterified cholesterol, phospholipids, and fatty acids into COS-7 cells. This lipid uptake is effected from three different lipid donor particles: mixed bile salt micelles, phospholipid small unilamellar vesicles, and trioleoylglycerol emulsions which are all likely to be present in the small intestine. Ezetimibe, a representative of a new class of drugs that inhibit intestinal cholesterol absorption, blocks SR-BI- and CD36-facilitated uptake of cholesterol into COS-7 cells.     

The cellular biology of scavenger receptor class B type I

The HDL receptor scavenger receptor class B type I plays an important role in meditating the uptake of HDL-derived cholesterol and cholesteryl ester in the liver and steroidogenic tissues. However, the mechanism by which scavenger receptor class B type I mediates selective cholesterol uptake is unclear. In hepatocytes scavenger receptor class B type I mediates the transcytosis of cholesterol into bile, appears to be expressed on both basolateral and apical membranes, and directly interacts with a PDZ domain containing protein that may modulate the activity of scavenger receptor class B type I. This suggests the involvement of scavenger receptor class B type I in higher order complexes in polarized cells. Scavenger receptor class B type I expression has been shown to alter plasma membrane cholesterol distribution and induce the formation of novel membrane structures, suggesting multiple roles for scavenger receptor class B type I in the cell. A close examination of scavenger receptor class B type I function in polarized cells may yield new insights into the mechanism of scavenger receptor class B type I-mediated HDL selective uptake and the effects of scavenger receptor class B type I on cellular cholesterol homeostasis.     

Drosophila ninaB and ninaD act outside of retina to produce rhodopsin chromophore

The Drosophila ninaB gene encodes a beta,beta-carotene-15,15'-oxygenase responsible for the centric cleavage of beta-carotene that produces the retinal chromophore of rhodopsin. The ninaD gene encodes a membrane receptor required for efficient use of beta-carotene. Despite their importance to the synthesis of visual pigment, we show that these genes are not active in the retina. Mosaic analysis shows that ninaB and ninaD are not required in the retina, and exclusive retinal expression of either gene, or both genes simultaneously, does not support rhodopsin biogenesis. In contrast, neuron-specific expression of ninaB and ninaD allows for rhodopsin biogenesis. Additional directed expression studies failed to identify other tissues supporting ninaB activity in rhodopsin biogenesis. These results show that nonretinal sites of NinaB beta,beta-carotene-15,15'-oxygenase activity, likely neurons of the central nervous system, are essential for production of the visual chromophore. Retinal or another C(20) retinoid, not members of the beta-carotene family of C(40) carotenoids, are supplied to photoreceptors for rhodopsin biogenesis.     

Photoprotective potential of lycopene,beta-carotene,vitamin E,vitamin C and carnosic acid in UVA-irradiated human skin fibroblasts

The photoprotective potential of the dietary antioxidants vitamin C, vitamin E, lycopene, beta-carotene, and the rosemary polyphenol, carnosic acid, was tested in human dermal fibroblasts exposed to ultraviolet-A (UVA) light. The carotenoids were prepared in special nanoparticle formulations together with vitamin C and/or vitamin E. Nanoparticle formulations, in contrast to dimethylsulphoxide, stablized lycopene in the cell culture medium and allowed efficient cellular uptake. The presence of vitamin E in the formulation further increased the stability and cellular uptake of lycopene. UVA irradiation of the human skin fibroblasts led to a 10-15-fold rise in metalloproteinase 1 (MMP-1) mRNA. This rise was suppressed in the presence of low microM concentrations of vitamin E, vitamin C, or carnosic acid but not with beta-carotene or lycopene. Indeed, in the presence of 0.5-1.0 microM beta-carotene or lycopene, the UVA-induced MMP-1 mRNA was further increased by 1.5-2-fold. This increase was totally suppressed when vitamin E was included in the nanoparticle formulation. Heme-oxygenase 1 (HO-1) mRNA expression was strongly induced by UVA irradiation but none of the antioxidants inhibited this effect at the concentrations used in this study. Indeed, beta-carotene or lycopene (0.5-1.0 microM) led to a further 1.5-fold rise in the UVA-induced HO-1 mRNA levels. In conclusion, vitamin C, vitamin E, and carnosic acid showed photoprotective potential. Lycopene and beta-carotene did not protect on their own but in the presence of vitamin E, their stability in culture was improved and the rise in MMP-1 mRNA expression was suppressed, suggesting a requirement for antioxidant protection of the carotenoids against formation of oxidative derivatives that can influence the cellular and molecular responses.     

Cellular mechanisms of vitamin E uptake: relevance in alpha-tocopherol metabolism and potential implications for disease

alpha-Tocopherol is an essential micronutrient involved in various oxidative stress-related processes. Because of its hydrophobic nature, alpha-tocopherol is transported in plasma lipoproteins, and the pathways involved in its cellular uptake are closely related to the lipoprotein metabolism. alpha-Tocopherol transfer from plasma to cells can occur by different mechanisms such as uptake facilitated by lipid transfer proteins and lipases, receptor-mediated lipoprotein endocytosis, and selective lipid uptake. Here we discuss recent progress in understanding the physiological and pathophysiological relevance of these different pathways for cellular uptake of vitamin E in vivo. This review is mainly focused on the role of the scavenger receptor class B type I (SR-BI) on alpha-tocopherol metabolism and its potential implications for disease conditions.     

Proteins involved in fat-soluble vitamin and carotenoid transport across the intestinal cells: New insights from the past decade

It is now well established that vitamins D, E, and K and carotenoids are not absorbed solely through passive diffusion. Broad-specificity membrane transporters such as SR-BI (scavenger receptor class B type I), CD36 (CD36 molecule), NPC1L1 (Niemann Pick C1-like 1) or ABCA1 (ATP-binding cassette A1) are involved in the uptake of these micronutrients from the lumen to the enterocyte cytosol and in their secretion into the bloodstream. Recently, the existence of efflux pathways from the enterocyte back to the lumen or from the bloodstream to the lumen, involving ABCB1 (P-glycoprotein/MDR1) or the ABCG5/ABCG8 complex, has also been evidenced for vitamins D and K. Surprisingly, no membrane proteins have been involved in dietary vitamin A uptake so far. After an overview of the metabolism of fat-soluble vitamins and carotenoids along the gastrointestinal tract (from the mouth to the colon where interactions with microbiota may occur), a focus is placed on the identified and candidate proteins participating in the apical uptake, intracellular transport, basolateral secretion and efflux back to the lumen of fat-soluble vitamins and carotenoids in enterocytes. This review also highlights the mechanisms that remain to be identified to fully unravel the pathways involved in fat-soluble vitamin and carotenoid intestinal absorption.     

Xanthophylls are preferentially taken up compared with beta-carotene by retinal cells via a SRBI-dependent mechanism

The purpose of this study was to investigate the mechanisms by which carotenoids [xanthophylls vs. beta-carotene(beta-C)] are taken up by retinal pigment epithelial (RPE) cells. The human RPE cell line, ARPE-19, was used. When ARPE-19 cells were fully differentiated (7-9 weeks), the xanthophylls lutein (LUT) and zeaxanthin (ZEA) were taken up by cells to an extent 2-fold higher than beta-C (P < 0.05). At 9 weeks, cellular uptakes were 1.6, 2.5, and 3.2%, respectively, for beta-C, LUT, and ZEA. Similar extents were observed when carotenoids were delivered in either Tween 40 or "chylomicrons" produced by Caco-2 cells. Differentiated ARPE-19 cells did not exhibit any detectable beta-C 15,15'-oxygenase activity or convert exogenous beta-C into vitamin A. When using specific antibodies against the lipid transporters cluster determinant 36 (CD36) and scavenger receptor class B type I (SR-BI), cellular uptake of beta-C and ZEA were significantly decreased (40-60%) with anti-SR-BI but not with anti-CD36. Small interfering RNA transfection for SR-BI led to marked knockdown of SR-BI protein expression (approximately 90%), which resulted in decreased beta-C and ZEA uptakes by 51% and 87%, respectively. Thus, the present data show that RPE cells preferentially take up xanthophylls versus the carotene by a process that appears to be entirely SR-BI-dependent for ZEA and partly so for beta-C. This mechanism may explain, in part, the preferential accumulation of xanthophylls in the macula of the retina.     

Biological actions of carotenoids

Of 600 carotenoids from natural sources that have been characterized, fewer than 10% serve as precursors of vitamin A. Many dietary carotenoids, both with and without provitamin A activity, are found in the blood and tissues of humans. beta-Carotene, the most nutritionally active carotenoid, comprises 15-30% of total serum carotenoids. Vitamin A is formed primarily by the oxygen-dependent central cleavage of beta-carotene and other provitamin A carotenoids. Several carotenoids show enhancement of the immune response, inhibition of mutagenesis, reduction of induced nuclear damage, and protection from various neoplastic events in cells, tissues, and whole animals. Carotenoids also protect against photo-induced tissue damage. Some carotenoids, including beta-carotene, quench highly reactive singlet oxygen under certain conditions and can block free radical-mediated reactions. In epidemiological studies, the intake of carotenoid-rich fruits and vegetables has been correlated with protection from some forms of cancer, particularly lung cancer. Similarly, serum beta-carotene levels have been associated with a decreased chance of developing lung cancer. It must be stressed, however, that these epidemiological associations do not show cause and effect. In this regard, long-term intervention trials with beta-carotene supplements are in progress. Whatever the results of these trials, carotenoids clearly show biological actions in animals distinct from their function as precursors of vitamin A.     

ASTER-B regulates mitochondrial carotenoid transport and homeostasis

The scavenger receptor class B type 1 (SR-B1) facilitates uptake of cholesterol and carotenoids into the plasma membrane (PM) of mammalian cells. Downstream of SR-B1, ASTER-B protein mediates the nonvesicular transport of cholesterol to mitochondria for steroidogenesis. Mitochondria also are the place for the processing of carotenoids into diapocarotenoids by β-carotene oxygenase-2. However, the role of these lipid transport proteins in carotenoid metabolism has not yet been established. Herein, we showed that the recombinant StART-like lipid-binding domain of ASTER-A and B preferentially binds oxygenated carotenoids such as zeaxanthin. We established a novel carotenoid uptake assay and demonstrated that ASTER-B expressing A549 cells transport zeaxanthin to mitochondria. In contrast, the pure hydrocarbon β-carotene is not transported to the organelles, consistent with its metabolic processing to vitamin A in the cytosol by β-carotene oxygenase-1. Depletion of the PM from cholesterol by methyl-β-cyclodextrin treatment enhanced zeaxanthin but not β-carotene transport to mitochondria. Loss-of-function assays by siRNA in A549 cells and the absence of zeaxanthin accumulation in mitochondria of ARPE19 cells confirmed the pivotal role of ASTER-B in this process. Together, our study in human cell lines established ASTER-B protein as key player in nonvesicular transport of zeaxanthin to mitochondria and elucidated the molecular basis of compartmentalization of the metabolism of nonprovitamin A and provitamin A carotenoids in mammalian cells.     

Scavenger receptors for oxidized and glycated proteins

Summary. Our present knowledge on chemically modified proteins and their receptor systems is originated from a proposal by Goldstein and Brown in 1979 for the receptor for acetylated LDL which is involved in foam cell formation, one of critical steps in atherogenesis. Subsequent extensive studies using oxidized LDL (OxLDL) as a representative ligand disclosed at least 11 different scavenger receptors which are collectively categorized as scavenger receptor family. Advanced glycation endproducts (AGE) and their receptor systems have been studied independently until recent findings that AGE-proteins are also recognized as active ligands by scavenger receptors including class A scavenger receptor (SR-A), class B scavenger receptors such as CD36 and SR-BI, type D scavenger receptor (LOX-1) and FEEL-1/FEEL-2. Three messages can be summarized from these experiments; (i) endocytic uptake of OxLDL and AGE-proteins by macrophages or macrophage-derived cells is mainly mediated by SR-A and CD36, which is an important step for foam cell formation in the early stage of atherosclerosis, (ii) selective uptake of cholesteryl esters of high density lipoprotein (HDL) mediated by SR-BI is inhibited by AGE-proteins, suggesting a potential pathological role of AGE in a HDL-mediated reverse cholesterol transport system, (iii) a novel scavenger receptor is involved in hepatic clearance of plasma OxLDL and AGE-proteins.     

Distinct mechanisms for OxLDL uptake and cellular trafficking by class B scavenger receptors CD36 and SR-BI

Modified forms of LDL, including oxidized low density lipoprotein (OxLDL), contribute to macrophage lipid accumulation in the vessel wall. Despite the pathophysiological importance of uptake pathways for OxLDL, the molecular details of OxLDL endocytosis by macrophages are not well understood. Studies in vitro demonstrate that the class B scavenger receptor CD36 mediates macrophage uptake and degradation of OxLDL. Although the closely related scavenger receptor class B type I (SR-BI) binds OxLDL with high affinity, evidence that SR-BI plays a role in OxLDL metabolism is lacking. In this study, we directly compared OxLDL uptake and degradation by CD36 and SR-BI. Our results indicate that although CD36 and SR-BI internalize OxLDL, SR-BI mediates significantly less OxLDL degradation. Endocytosis of OxLDL by both SR-BI and CD36 is independent of caveolae, microtubules, and actin cytoskeleton. However, OxLDL uptake by CD36, but not SR-BI, is dependent on dynamin. The analysis of chimeric SR-BI/CD36 receptors shows that the CD36 C-terminal cytoplasmic tail is necessary and sufficient for dynamin-dependent OxLDL internalization by class B scavenger receptors. These findings indicate that different mechanisms are involved in OxLDL uptake by SR-BI and CD36, which may segregate these two structurally homologous receptors at the cell surface, leading to differences in intracellular trafficking and degradation.     

Provitamin A conversion to retinal via the beta,beta-carotene-15,15'-oxygenase (bcox) is essential for pattern formation and differentiation during zebrafish embryogenesis

The egg yolk of vertebrates contains carotenoids, which account for its characteristic yellow color in some species. Such plant-derived compounds, e.g. beta-carotene, serve as the natural precursors (provitamins) of vitamin A, which is indispensable for chordate development. As egg yolk also contains stored vitamin A, carotenoids have so far been solely discussed as pigments for the coloration of the offspring. Based on our recent molecular identification of the enzyme catalyzing provitamin A conversion to vitamin A, we address a possible role of provitamin A during zebrafish (Danio rerio) development. We cloned the zebrafish gene encoding the vitamin A-forming enzyme, a beta,beta-carotene-15,15'-oxygenase. Analysis of its mRNA expression revealed that it is under complex spatial and temporal control during development. Targeted gene knockdown using the morpholino antisense oligonucleotide technique indicated a vital role of the provitamin A-converting enzyme. Morpholino-injected embryos developed a morphological phenotype that included severe malformation of the eyes, the craniofacial skeleton and pectoral fins, as well as reduced pigmentation. Analyses of gene expression changes in the morphants revealed that distinct retinoic acid-dependent developmental processes are impaired, such as patterning of the hindbrain and differentiation of hindbrain neurons, differentiation of neural crest derivatives (including the craniofacial skeleton), and the establishment of the ventral retina. Our data provide strong evidence that, for several developmental processes, retinoic acid generation depends on local de novo formation of retinal from provitamin A via the carotene oxygenase, revealing an unexpected, essential role for carotenoids in embryonic development.     

Proteins involved in uptake, intracellular transport and basolateral secretion of fat-soluble vitamins and carotenoids by mammalian enterocytes

Our understanding of the molecular mechanisms responsible for fat-soluble vitamin uptake and transport at the intestinal level has advanced considerably over the past decade. On one hand, it has long been considered that vitamin D and E as well as β-carotene (the main provitamin A carotenoid in human diet) were absorbed by a passive diffusion process, although this could not explain the broad inter-individual variability in the absorption efficiency of these molecules. On the other hand, it was assumed that preformed vitamin A (retinol) and vitamin K1 (phylloquinone) absorption occurred via energy-dependent processes, but the transporters involved have not yet been identified. The recent discovery of intestinal proteins able to facilitate vitamin E and carotenoid uptake and secretion by the enterocyte has spurred renewed interest in studying the fundamental mechanisms involved in the absorption of these micronutrients. The proteins identified so far are cholesterol transporters such as SR-BI (scavenger receptor class B type I), CD36 (cluster determinant 36), NPC1L1 (Niemann–Pick C1-like 1) or ABCA1 (ATP-Binding Cassette A1) displaying a broad substrate specificity, but it is likely that other membrane proteins are also involved. After overviewing the metabolism of fat-soluble vitamins and carotenoids in the human upper gastrointestinal lumen, we will focus on the putative or identified proteins participating in the intestinal uptake, intracellular transport and basolateral secretion of these fat-soluble vitamins and carotenoids, and outline the uncertainties that need to be explored in the future. Identifying the proteins involved in intestinal uptake and transport of fat-soluble vitamins and carotenoids across the enterocyte is of great importance, especially as some of them are already targets for the development of drugs able to slow cholesterol absorption. Indeed, these drugs may also interfere with lipid vitamin uptake. A better understanding of the molecular mechanisms involved in fat-soluble vitamin and carotenoid absorption is a priority to better optimize their bioavailability.     

Intestinal absorption and metabolism of carotenoids: insights from cell culture

Cell culture models are useful for studying intestinal absorption and metabolism of carotenoids. The human intestinal cell line, Caco-2, has been the most widely used model for these studies. The PF11 and TC7 clones of Caco-2 exhibit beta-carotene-15,15'-oxygenase activity, a key enzyme in the conversion of carotenoids to vitamin A. Studies on the recent cloning of this enzyme are discussed. An in vitro cell culture system used to study intestinal absorption of carotenoids is presented. Under conditions mimicking the postprandial state, Caco-2 cells on membranes take up carotenoids and secrete them incorporated into chylomicrons. Both the cellular uptake and secretion of beta-carotene are saturable, concentration-dependent processes. The selective absorption of all-trans beta-carotene versus its cis isomers, the differential absorption of individual carotenoids, and the specific interactions between carotenoids during their absorption are discussed. The participation of a specific epithelial transporter in the intestinal absorption of carotenoids is proposed.     

TGF-beta1 downregulates CD36 and scavenger receptor A but upregulates LOX-1 in human macrophages

Transforming growth factor-beta1 (TGF-beta1), a key cytokine for control of cell growth, extracellular matrix formation, and inflammation control, is secreted by many cells present in the arteriosclerotic plaque. Lipid accumulation in the vessel wall is regarded as an early step in atherogenesis and depends on uptake of modified low-density lipoprotein (LDL) by macrophages through scavenger receptors and their transformation into foam cells. Prominent members of the scavenger receptor family are the class A type I and II receptors (ScR-A), the class B receptor CD36, and the recently detected lectin-like oxidized LDL receptor-1 (LOX-1), which, unlike the native LDL receptor (LDL-R), are not feedback controlled. CD36 is responsible for >50% of modified LDL uptake into human monocyte-derived macrophages. We therefore studied whether TGF-beta1 influences expression and function of ScR-A, CD36, and LOX-1 in monocytes using RT-PCR and flow cytometry. Total uptake of oxidized LDL by monocytoid cells, reflecting the combined function of all scavenger receptors, was significantly reduced by TGF-beta1. At initially low picomolar concentrations, TGF-beta1 decreased CD36 mRNA and protein surface expression and ScR-A mRNA levels in the human monocytic cell line THP-1 and in freshly isolated and cultivated human monocytes, whereas LOX-1 mRNA was increased. Expression of LDL-R and beta-actin was not affected by TGF-beta1. In conclusion, depression of scavenger receptor function in monocytes by TGF-beta1 in low concentrations reduces foam cell formation. Together with matrix control by TGF-beta1, this may be important for atherogenesis and plaque stabilization.     

Evidence for a Trypanosoma brucei lipoprotein scavenger receptor

African trypanosomes are lipid auxotrophs that live in the bloodstream of their human and animal hosts. Trypanosomes require lipoproteins in addition to other serum components in order to multiply under axenic culture conditions. Delipidation of the lipoproteins abrogates their capacity to support trypanosome growth. Both major classes of serum lipoproteins, LDL and HDL, are primary sources of lipids, delivering cholesterol esters, cholesterol, and phospholipids to trypanosomes. We show evidence for the existence of a trypanosome lipoprotein scavenger receptor, which facilitates the endocytosis of both native and modified lipoproteins, including HDL and LDL. This lipoprotein scavenger receptor also exhibits selective lipid uptake, whereby the uptake of the lipid components of the lipoprotein exceeds that of the protein components. Trypanosome lytic factor (TLF1), an unusual HDL found in human serum that protects from infection by lysing Trypanosoma brucei brucei, is also bound and endocytosed by this lipoprotein scavenger receptor. HDL and LDL compete for the binding and uptake of TLF1 and thereby attenuate the trypanosome lysis mediated by TLF1. We also show that a mammalian scavenger receptor facilitates lipid uptake from TLF1 in a manner similar to the trypanosome scavenger receptor. Based on these results we propose that HDL, LDL, and TLF1 are all bound and taken up by a lipoprotein scavenger receptor, which may constitute the parasite's major pathway mediating the uptake of essential lipids.