Carotenoid Transport Research Articles

A mutation in the promoter of the chicken β,β-carotene 15,15′-monooxygenase 1 gene alters xanthophyll metabolism through a selective effect on its mRNA abundance in the breast muscle1

A polymorphism in the promoter of the β,β-carotene 15,15'-monooxygenase 1 (BCMO1) gene recently was identified in an experimental cross between 2 chicken lines divergently selected on growth rate and found to be associated with variations in the yellow color of the breast meat. In this study, the effects of the polymorphism on several aspects of carotenoid metabolism were evaluated in chickens sharing the same genetic background except for their genotype at the BCMO1 locus. We confirmed that BCMO1 mRNA abundance varied (P < 0.001) between the 2 homozygous genotypes (GG << AA) and in the pectoralis major muscle. By contrast, BCMO1 mRNA expression was not affected (P > 0.05) by the polymorphism in the duodenum, liver, or sartorius muscle. The breast meat of GG chickens was more (P < 0.001) yellow and richer in lutein (P < 0.01) and zeaxanthin (P < 0.05) compared to that of AA chickens whereas these variables did not differ (P > 0.05) in the other tissues tested. The GG were also characterized by reduced (P < 0.01) plasma lutein and zeaxanthin concentrations than AA without affecting plasma and tissue content of fat-soluble vitamins A and E. As lutein and zeaxanthin are usually not considered as substrates of the BCMO1 enzyme, the impact of BCMO1 polymorphism on the activity of other genes involved in carotenoid transport (SCARB1 and CD36 encoding the scavenger receptor class B type I and the cluster determinant 36, respectively) and metabolism (BCDO2 encoding β,β-carotene 9',10'-dioxygenase 2) was evaluated. The BCMO1 polymorphism did not affect mRNA abundance of BCDO2, SCARB1, or CD36, regardless of tissue considered. Taken together, these results indicated that a genetic variant of BCMO1 specifically changes lutein and zeaxanthin content in the chicken plasma and breast muscle without impairing vitamin A and E metabolism.

Prawn lipocalin: characterization of a color shift induced by gene knockdown and ligand binding assay

The lipocalin family of proteins functions in the transport of steroids, carotenoids, retinoids, and other small hydrophobic molecules. Recently, a lipocalin (MrLC) was isolated from the prawn Macrobrachium rosenbergii and its expression varied with the molting cycle. In this study, knockdown of the MrLC gene by RNA interference (RNAi) was performed and resulted in a shift in body color from blue to orangish red over the entire carapace. By immune-gold electron microscopy, MrLC was found to co-localize with the lipid droplets in subepidermal adiose tissue that were found to be decreased dramatically in MrLC knockdown prawns, in which a reduction in relative fat content was also quantified. Furthermore, MrLC was found to specifically bind astaxanthin and molt hormone (20-hydroxyecdysone) in both in vitro ligand binding assay and in vivo native ligand detection. These results suggested that MrLC plays roles in the regulation of coloration through its association with astaxanthin and may also be involved in the regulation of molting in crustacean.

Free Radical Exposure Creates Paler Carotenoid-Based Ornaments: A Possible Interaction in the Expression of Black and Red Traits

Oxidative stress could be a key selective force shaping the expression of colored traits produced by the primary animal pigments in integuments: carotenoids and melanins. However, the impact of oxidative stress on melanic ornaments has only recently been explored, whereas its role in the expression of carotenoid-based traits is not fully understood. An interesting study case is that of those animal species simultaneously expressing both kinds of ornaments, such as the red-legged partridge (Alectoris rufa). In this bird, individuals exposed to an exogenous source of free radicals (diquat) during their development produced larger eumelanin-based (black) plumage traits than controls. Here, we show that the same red-legged partridges exposed to diquat simultaneously developed paler carotenoid-based ornaments (red beak and eye rings), and carried lower circulating carotenoid levels as well as lower levels of some lipids involved in carotenoid transport in the bloodstream (i.e., cholesterol). Moreover, partridges treated with a hormone that stimulates eumelanin production (i.e., alpha-melanocyte-stimulating hormone) also increased blood carotenoid levels, but this effect was not mirrored in the expression of carotenoid-based traits. The redness of carotenoid-based ornaments and the size of a conspicuous eumelanic trait (the black bib) were negatively correlated in control birds, suggesting a physiological trade-off during development. These findings contradict recent studies questioning the sensitivity of carotenoids to oxidative stress. Nonetheless, the impact of free radicals on plasma carotenoids seems to be partially mediated by changes in cholesterol metabolism, and not by direct carotenoid destruction/consumption. The results highlight the capacity of oxidative stress to create multiple phenotypes during development through differential effects on carotenoids and melanins, raising questions about evolutionary constraints involved in the production of multiple ornaments by the same organism.

Bioaccessibility, Uptake, and Transport of Carotenoids from Peppers (Capsicum Spp.) Using the Coupled in Vitro Digestion and Human Intestinal Caco-2 Cell Model

Spanish bell peppers (Capsicum annuum L.) and chili peppers sourced from Kenya and Turkey were analyzed for their carotenoid content, bioaccessibility, and bioavailability. The order of total carotenoid content in peppers and their respective micelles was red > green > yellow. In terms of cellular carotenoid transport as a percentage of original food and micelle content, the order was yellow peppers > green > red; however, the opposite trend was seen for the actual amount of total carotenoids transported by Caco-2 cells. Although lutein was generally the most abundant carotenoid in the micelles (496.3-1565.7 microg 100 g(-1)), cellular uptake and transport of beta-carotene were the highest, 8.3-31.6 and 16.8-42.7%, respectively. Hence, the actual amount of carotenoids present in the original food and respective micelles seems to reflect the amount transported by Caco-2 cells. Therefore, color influenced the carotenoid profile, bioaccessibility, and bioavailability of carotenoids rather than pepper type.

A CD36-related Transmembrane Protein Is Coordinated with an Intracellular Lipid-binding Protein in Selective Carotenoid Transport for Cocoon Coloration

The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.

Competitive inhibition of carotenoid transport and tissue concentrations by high dose supplements of lutein, zeaxanthin and beta-carotene

Carotenoids may interact differently in their absorption and transport in animals and humans. The simultaneous administration of large amounts of lutein, zeaxanthin and beta carotene would affect not only plasma values but also their concentrations in the retina and other tissues. In this study, we investigated the transport, distribution and interactions of lutein, zeaxanthin and beta-carotene in the plasma, retina and other tissues of chicks fed supplements rich in lutein, zeaxanthin or beta-carotene. Newly hatched male Leghorn chicks were randomly assigned to ten groups. One group provided baseline data (1-day-old group). The other groups were fed one of the following six diets for 14 or 28 days: high lutein diet; high zeaxanthin diet; three high beta-carotene supplemented diets and the control diet. Plasma and tissues including retina were analyzed for lutein and zeaxanthin and beta-carotene at baseline and at 14 and 28 days. All tissues had increased concentrations of lutein after the high lutein diet and had increased concentrations of zeaxanthin after the high zeaxanthin diet. After 28 days, the retinal concentrations of lutein and zeaxanthin in the chicks supplemented with lutein (27.2 mg/kg diet) and zeaxanthin (15.3 mg/kg diet) increased 128 and 116%, respectively, compared to the retinas of chicks fed the control diet (lutein 5.2 mg/kg and zeaxanthin 1.7 mg/kg). Lutein was decreased in plasma and other non-retinal tissues when the diet was supplemented with zeaxanthin; likewise, zeaxanthin was decreased in plasma and non-retinal tissues after the lutein supplement. Zeaxanthin increased in the retina after the high lutein supplement, and retinal lutein was maintained after the high zeaxanthin supplement. The high beta-carotene supplement increased the beta-carotene content of plasma and liver very little, and beta-carotene was not found in any other tissue in the chick, including the retina. More importantly, beta-carotene decreased the concentrations of both lutein and zeaxanthin in the plasma and most tissues, including the retina. High dose dietary supplementation of a single carotenoid may alter the assimilation of other carotenoids. The retina appears to have the capacity to preserve accumulation of lutein and zeaxanthin, but this capacity is diminished when intake of beta-carotene is high.

Purification and Partial Characterization of a Lutein-Binding Protein from Human Retina

Dietary intake of lutein and zeaxanthin appears to be advantageous for protecting human retinal and macular tissues from degenerative disorders such as age-related macular degeneration. Selective concentration of just two of the many dietary carotenoids suggests that uptake and transport of these xanthophyll carotenoids into the human foveal region are mediated by specific xanthophyll-binding proteins such as GSTP1 which has previously been identified as the zeaxanthin-binding protein of the primate macula. Here, a membrane-associated human retinal lutein-binding protein (HR-LBP) was purified from human peripheral retina using ion-exchange chromatography followed by size-exclusion chromatography. After attaining 83-fold enrichment of HR-LBP, this protein exhibited a significant bathochromic shift of approximately 90 nm in association with lutein, and equilibrium binding studies demonstrated saturable, specific binding toward lutein with a K(D) of 0.45 muM. Examination for cross-reactivity with antibodies raised against known lutein-binding proteins from other organisms revealed consistent labeling of a major protein band of purified HR-LBP at approximately 29 kDa with an antibody raised against silkworm (Bombyx mori) carotenoid-binding protein (CBP), a member of steroidogenic acute regulatory (StAR) protein family with significant homology to many human StAR proteins. Immunolocalization with antibodies directed against either CBP or GSTP1 showed specific labeling of rod and cone inner segments, especially in the mitochondria-rich ellipsoid region. There was also strong labeling of the outer plexiform (Henle fiber) layer with anti-GSTP1. Such localizations compare favorably with the distribution of macular carotenoids as revealed by resonance Raman microscopy. Our results suggest that HR-LBP may facilitate lutein's localization to a region of the cell subject to considerable oxidative stress.

Global shape and pH stability of ovorubin, an oligomeric protein from the eggs of Pomacea canaliculata

Ovorubin, a 300-kDa thermostable oligomer, is the major egg protein from the perivitellin fluid that surrounds the embryos of the apple snail Pomacea canaliculata. It plays essential roles in embryo development, including transport and protection of carotenoids, protease inhibition, photoprotection, storage, and nourishment. Here, we report ovorubin dimensions and global shape, and test the role of electrostatic interactions in conformational stability by analyzing the effects of pH, using small-angle X-ray scattering (SAXS), transmission electron microscopy, CD, and fluorescence and absorption spectroscopy. Analysis of SAXS data shows that ovorubin is an anisometric particle with a major axis of 130 A and a minor one varying between 63 and 76 A. The particle shape was not significantly affected by the absence of the cofactor astaxanthin. The 3D model presented here is the first for an invertebrate egg carotenoprotein. The quaternary structure is stable over a wide pH range (4.5-12.0). At a pH between 2.0 and 4.0, a reduction in the gyration radius and a loss of tertiary structure are observed, although astaxanthin binding is not affected and only minor alterations in secondary structure are observed. In vitro pepsin digestion indicates that ovorubin is resistant to this protease action. The high stability over a considerable pH range and against pepsin, together with the capacity to bear temperatures > 95 degrees C, reinforces the idea that ovorubin is tailored to withstand a wide variety of conditions in order to play its key role in embryo protection during development.

Expression of Genes Associated with Retinoid Metabolism in the Trout Ovarian Follicle1

In vertebrates producing oviparous eggs, retinoids and their precursor molecules need to be deposited in oocytes during vitellogenesis. While most studies focus on the transport of retinoids and carotenoids formed outside the fish ovary and their deposition within the developing oocyte, recent investigations in mammalian species suggest the ovary is an important site for retinoid and carotenoid metabolism. Therefore, we investigated the expression of six genes (bcmo1, bcdo2, rbp1, lrat, rbp4, and stra6) associated with retinoids and carotenoids in juvenile and adult trout ovaries. Except for bcdo2, these genes were expressed in the ovary. Expression of stra6 was detected in the ovary but not in the liver. Gene expression levels of bcmo1 and stra6 were significantly higher in juvenile ovaries, in contrast to those of rbp1, rbp4, and lrat, which were similar in all tested ovarian stages. The mean values of the relative mRNA levels of the tested genes differed between the ovary and the liver. Gene transcripts of rbp4 and bcmo1 were identified by in situ hybridization in the theca layer, and all five genes were expressed in the granulosa, stromal cells, and only the early vitellogenic oocyte. The occurrence of retinol-binding protein in the theca and granulosa cells and within oocytes at all developmental stages was revealed by immunocytochemistry. These results indicate that ovarian cells express genes putatively associated with cleavage of beta-carotene, storage and mobilization of retinyl-esters, and of retinol-binding protein synthesis, suggesting a novel pathway for providing retinoids and carotenoids to developing fish ovarian follicles.

In vitro screening of relative bioaccessibility of carotenoids from foods.

Carotenoids are lipophilic pigments in plant foods that are of particular interest as precursors of vitamin A, a nutrient required for vision, cell differentiation, and the immune system. In order to mediate such activities, carotenoids and their metabolites must be absorbed for delivery to tissues. Unlike many other dietary lipids, the efficiency of carotenoid absorption is typically inefficient, being affected by food matrix, style of processing, other dietary components, and nutritional and physiological status. Thus, reliable prediction of carotenoid bioavailability is problematic. We have developed a relatively simple and cost effective procedure to study the potential bioavailability, i.e., the bioaccessibility, of carotenoids. The method involves simulated oral, gastric and small intestinal digestion of test samples to access the efficiency of incorporation into micelles, an obligatory step for absorption of lipophilic compounds. The model can be further expanded by adding micelles generated during small intestinal phase of digestion to monolayers of Caco-2 human intestinal epithelial cells to investigate apical uptake, cellular metabolism and transepithelial transport of carotenoids. Recent work by Borel and associates has demonstrated that the relative bioaccessibility of carotenoids observed in vitro is highly correlated with in vivo observations and results from bioavailability trials with human subjects. Results from recent studies using the in vitro model to screen relative bioaccessibility of beta-carotene in various cultivars of cassava, impact of amount and types of fatty acyl groups in triglycerides on micellarization of carotenoids, and the mechanism of digestion and intestinal cell uptake of xanthophyll esters are presented.

Mechanisms of provitamin A (carotenoid) and vitamin A (retinol) transport into and out of intestinal Caco-2 cells

The purpose of this study was to compare the mechanisms of intestinal retinol (ROL) and carotenoid transport. When differentiated Caco-2 cells were incubated with ROL for varying times, cellular ROL plateaued within 2 h, whereas retinyl ester (RE) formation increased continuously. ROL and RE efflux into basolateral medium (BM) increased linearly with time, ROL in the nonlipoprotein fraction and REs in chylomicrons (CMs). In contrast to carotenoids, ROL uptake was proportional to ROL concentration (0.5-110 microM). ROL efflux into BM occurred via two processes: a) a saturable process at low concentrations (<10 microM) and b) a nonsaturable process at higher concentrations. When ROL-loaded cells were maintained on retinoid-free medium, free ROL, but not REs, was secreted into BM. Glyburide significantly reduced ROL efflux but not ROL uptake. Inhibition of ABCA1 protein expression by small interfering RNAs decreased ROL efflux but not carotenoid efflux. Scavenger receptor class B type I (SR-BI) inhibition did not affect ROL transport but decreased carotenoid uptake. The present data suggest that a) ROL enters intestinal cells by diffusion, b) ROL efflux is partly facilitated, probably by the basolateral transporter ABCA1, and c) newly synthesized REs, but not preformed esters, are incorporated into CM and secreted. In contrast to ROL transport, carotenoid uptake is mediated by the apical transporter SR-BI, and carotenoid efflux occurs exclusively via their secretion in CM.

Nutritional factors and progenitor cell differentiation

Knowledge about the biological role of carotenoids is still very poor because of the bioaviability of carotenoids is complicated. Multiple factors influence tissues ability to: absorb, transport, metabolise and storage of carotenoids. Beta-carotene (BC) as well as fatty acids coming from food act as a source of energy and a regulator of metabolic processes [1–3]. About 10% of characterized natural carotenoids can be metabolized in human tissues to vitamin A, the precursor of retinoid acid (RA). Different type of carotenoids are present in human tissues and function as: free radical scavengers, immunomodulators, enhancers of the gap junction proteins, apoptosis regulators for many cell types, cancer preventive agents [4–9] Angiogenesis is the important process for the ischemic tissue growth and remodeling including the cancer as well as adipose tissue [10,11]. The main result of presented study demonstrated chemotactic activity of BC in vitro in relation to observed changes in gene expression in human endothelial cells and human umbilical cord blood originated endothelial progenitor cells.

Carotenoid silk coloration is controlled by a carotenoid-binding protein, a product of the Yellow blood gene

Mechanisms for the uptake and transport of carotenoids, essential nutrients for humans, are not well understood in any animal system. The Y (Yellow blood) gene, a critical cocoon color determinant in the silkworm Bombyx mori, controls the uptake of carotenoids into the intestinal mucosa and the silk gland. Here we provide evidence that the Y gene corresponds to the intracellular carotenoid-binding protein (CBP) gene. In the Y recessive strain, the absence of an exon, likely due to an incorrect mRNA splicing caused by a transposon-associated genomic deletion, generates a nonfunctional CBP mRNA, resulting in colorless hemolymph and white cocoons. Enhancement of carotenoid uptake and coloration of the white cocoon was achieved by germ-line transformation with the CBP gene. This study demonstrates the existence of a genetically facilitated intracellular process beyond passive diffusion for carotenoid uptake in the animal phyla, and paves the way for modulating silk color and lipid content through genetic engineering.

Effect of dietary lutein and zeaxanthin on plasma carotenoids and their transport in lipoproteins in age-related macular degeneration

Background:Low dietary intakes and low plasma concentrations of lutein and zeaxanthin are associated with an increased risk of age-related macular degeneration (AMD). No studies have challenged AMD patients with a diet high in lutein and zeaxanthin.Objective:The objective was to examine the effect of diets low or high in lutein and zeaxanthin on plasma carotenoids and their transport in AMD patients.Design:Seven AMD patients and 5 control subjects were fed a low-lutein, low-zeaxanthin diet (≈1.1 mg/d) for 2 wk, which was followed by a high-lutein, high-zeaxanthin diet (≈11 mg/d) for 4 wk. Ten subjects continued the diet for 8 wk. Plasma and lipoprotein carotenoids were measured by HPLC.Results:The high-lutein, high-zeaxanthin diet resulted in 2- to 3-fold increases in plasma concentrations of lutein and zeaxanthin and other carotenoids, except lycopene, in the AMD patients and the control subjects. With this diet, 52% of the lutein and 44% of the zeaxanthin were transported by HDL; ≈22% of lutein and zeaxanthin was transported by LDL. Only 20–25% of α-carotene, β-carotene, and lycopene was transported by HDL; 50–57% was transported by LDL.Conclusions:The AMD patients and control subjects responded similarly to a diet high in lutein and zeaxanthin; plasma carotenoid concentrations increased greatly in both groups, and the transport of carotenoids by lipoproteins was not significantly different between the groups. This finding suggests that abnormalities in the metabolism of lutein and zeaxanthin in AMD may reside in the uptake of lutein and zeaxanthin from the plasma and transport into the retina.

Astaxanthin binding and structural stability of the apple snail carotenoprotein ovorubin

Ovorubin (OR) is the major perivitellin of the eggs of Pomacea canaliculata. The astaxanthin (ASX) binding and structural stability of OR were investigated by fluorescence spectroscopy and circular dichroism (CD). The apo-OR (without astaxanthin) shows a single, high affinity binding site for ASX ( K D = 0.5 μM). The quenching of tryptophan fluorescence by ASX indicates that about 22% are near the carotenoid-binding site in a non-polar environment, as indicated by tryptophan resonance energy transfer to the ligand. Secondary structure (α + β) was virtually not affected by cofactor removal. Holo-OR shows unusually high thermal stability. The removal of ASX does not affect the thermal or chemical stability of the quaternary structure. In conclusion, although subtle changes were observed, ASX is not essential for OR stability, unlike most invertebrate carotenoproteins. This supports the idea that OR plays an important physiological role in the storage, transport and protection of carotenoids during snail embryogenesis.

Intestinal absorption of dietary carotenoids

The assessment of carotenoid bioavailability has long been hampered by the limited knowledge of their absorption mechanisms. However, recent reports have elucidated important aspects of carotenoid digestion and absorption. Disruption of food matrix and increasing amounts of fat seem to enhance the absorption of carotenes to a larger extent than that of xanthophylls. Comparing different carotenoid species, xanthophylls seem to be more easily released from the food matrix and more efficiently micellized than the carotenes. On the other hand, carotenes are more efficiently taken up by the enterocytes. However, carotenoid emulsification and micellization steps are largely affected by the food matrix and dietary components, being the main determinant of carotenoid bioavailability from foodstuffs. Although the intestinal uptake of carotenoids has been thought to occur by simple diffusion, recent studies reported the existence of receptor-mediated transport of carotenoids in enterocytes. Comparisons between the intestinal absorption of a wide array of carotenoids would be useful to elucidate the absorption mechanism of each carotenoid species, in view of the recent indications that intestinal carotenoid uptake may involve the scavenger receptor class B type I and possibly other epithelial transporters. The unraveling of the whole mechanism underlying the absorption of carotenoids will be the challenge for future studies.

Carotenoid Transport Is Decreased and Expression of the Lipid Transporters SR-BI, NPC1L1, and ABCA1 Is Downregulated in Caco-2 Cells Treated with Ezetimibe

Data suggest that intestinal carotenoid absorption is a facilitated process. The present study was conducted to determine whether carotenoids and cholesterol share common pathways (transporters) for their intestinal absorption. Differentiated Caco-2 cells on membranes were incubated (16 h) with a carotenoid (1 micromol/L) with or without ezetimibe (EZ; Zetia, an inhibitor of cholesterol transport), and with or without antibodies against the receptors, cluster determinant 36 (CD36) and scavenger receptor class B, type I (SR-BI). Carotenoid transport in Caco-2 cells (cellular uptake + secretion) was decreased by EZ (10 mg/L) as follows: beta-carotene approximately alpha-carotene (50% inhibition) >> beta-cryptoxanthin approximately lycopene (20%) >> lutein:zeaxanthin (1:1) (7%). EZ reduced cholesterol transport by 31%, but not retinol transport. beta-Carotene transport was also inhibited by anti-SR-BI, but not by anti-CD36. The inhibitory effects of EZ and anti-SR-BI on beta-carotene transport were additive, indicating that they may have different targets. Finally, differentiated Caco-2 cells treated with EZ showed a significant decrease in mRNA expression for the surface receptors SR-BI, Niemann-Pick type C1 Like 1 protein (NPC1L1), and ATP-binding cassette transporter, subfamily A (ABCA1) and for the nuclear receptors retinoid acid receptor (RAR)gamma, sterol-regulatory element binding proteins (SREBP)-1 and -2, and liver X receptor (LXR)beta as assessed by real-time PCR analysis. The data indicate that 1) EZ is an inhibitor of carotenoid transport, an effect that decreases with increasing polarity of the carotenoid molecule, 2) SR-BI is involved in carotenoid transport, and 3) EZ may act, not only by interacting physically with cholesterol transporters as previously suggested, but also by downregulating expression of these proteins. The cellular uptake and efflux of carotenoids, like that of cholesterol, likely involve more than one transporter.

Diet, Plasma Carotenoids, and Sexual Coloration in the Zebra Finch (Taeniopygia Guttata)

Abstract Carotenoid-based colors serve important sexual-signaling functions in many animals, but the proximate factor(s) underlying their expression has sparked controversy. In particular, the relative contributions of dietary and physiological mechanisms have been questioned of late. However, no studies have concurrently quantified levels of food intake or pigment processing in any species to examine the comparative effects of pigment acquisition and use on integumetary coloration. Here, we studied within- and between-sex patterns of food intake and plasma pigment circulation in the Zebra Finch (Taeniopygia guttata) to assess how sexually dichromatic, carotenoid-based bill pigmentation serves as an indicator of pigment access in the diet and carotenoid transport through the bloodstream. First, in a food-choice study, we found that males and females did not consume different types or amounts of food, despite dramatic sex differences in bill coloration. Similarly, variability in carotenoid-based bill pigmentation within each sex was uncoupled from levels of food consumption. Next, we used high-performance liquid-chromatography (HPLC) to quantify the types and amounts of carotenoids circulating through blood. Male and female Zebra Finches circulated the same four major carotenoid pigments in blood plasma (lutein, zeaxanthin, anhydrolutein, and β-cryptoxanthin), but males circulated a significantly higher concentration of plasma carotenoids than did females. Within both sexes, individuals that circulated more carotenoid pigments displayed more brightly colored bills. In sum, these results suggest that physiological factors such as pigment transport may play a more important role in shaping variability in carotenoid-based bill coloration in this species than does diet. Future studies should be aimed at identifying the proximate determinants of plasma carotenoid circulation in these birds as well as how circulated pigments are used to produce maximum color displays.

DIET, PLASMA CAROTENOIDS, AND SEXUAL COLORATION IN THE ZEBRA FINCH (TAENIOPYGIA GUTTATA)

Abstract Carotenoid-based colors serve important sexual-signaling functions in many animals, but the proximate factor(s) underlying their expression has sparked controversy. In particular, the relative contributions of dietary and physiological mechanisms have been questioned of late. However, no studies have concurrently quantified levels of food intake or pigment processing in any species to examine the comparative effects of pigment acquisition and use on integumetary coloration. Here, we studied within- and between-sex patterns of food intake and plasma pigment circulation in the Zebra Finch (Taeniopygia guttata) to assess how sexually dichromatic, carotenoid-based bill pigmentation serves as an indicator of pigment access in the diet and carotenoid transport through the bloodstream. First, in a food-choice study, we found that males and females did not consume different types or amounts of food, despite dramatic sex differences in bill coloration. Similarly, variability in carotenoid-based bill pigme...

Isolation, Characterization, and cDNA Sequence of a Carotenoid Binding Protein from the Silk Gland of Bombyx mori Larvae

A carotenoid binding protein (CBP) has been isolated from the silk glands of Bombyx mori larvae. The protein has an apparent molecular mass of 33 kDa and binds carotenoids in a 1:1 molar ratio. Lutein accounts for 90% of the bound carotenoids, whereas alpha-carotene and beta-carotene are minor components. Immunological analysis demonstrated the presence of CBP only in the yellow-colored tissues of the silk gland, midgut, testis, and ovary. Several phenotypes of B. mori mutants linked to carotenoid transport have been utilized to characterize CBP. The Y (yellow hemolymph) gene controls uptake of carotenoids from the midgut lumen into the midgut epithelium, and larvae with the +(Y) gene lack this property. Immunoblotting analysis confirmed the presence of CBP in mutants with the dominant Y gene only. Immunohistochemistry verified the localization of CBP in the villi of the midgut epithelium, indicating that CBP might be involved in absorption of carotenoids. A cDNA clone for CBP encoding a protein of 297 amino acids has been isolated from the B. mori silk gland cDNA library. The deduced amino acid sequence revealed that CBP is a novel member of the steroidogenic acute regulatory (StAR) protein family with its unique structural feature of a StAR-related lipid transfer domain, known to aid in lipid transfer and recognition. Lutein-binding capacity of the recombinant CBP (rCBP) determined by incubating rCBP with lutein followed by immunoprecipitation using anti-CBP IgG conjugated to protein A-Sepharose, demonstrated the formation of a lutein-rCBP complex. Sequence analyses coupled with binding specificity suggest that CBP is a new member of the StAR protein family that binds carotenoids rather than cholesterol.