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Background: Nonalcoholic fatty liver disease is positively associated with obesity and cardiovascular disease risk. Apo-10'-lycopenoic acid (APO10LA), a potential oxidation product of apo-10'-lycopenal that is generated endogenously by β-carotene-9',10'-oxygenase (BCO2) cleavage of lycopene, inhibited hepatic steatosis in BCO2-expressing mice. Objective: The present study evaluated lycopene and APO10LA effects on hepatic steatosis in mice without BCO2 expression. Methods: Male and female BCO2-knockout (BCO2-KO) mice were fed a high saturated fat diet (HSFD) with or without APO10LA (10 mg/kg diet) or lycopene (100 mg/kg diet) for 12 wk. Results: Lycopene or APO10LA supplementation reduced hepatic steatosis incidence (78% and 72%, respectively) and severity in BCO2-KO male mice. Female mice did not develop steatosis, had greater hepatic total cholesterol (3.06 vs. 2.31 mg/g tissue) and cholesteryl ester (1.58 vs. 0.86 mg/g tissue), but had lower plasma triglyceride (TG) (229 vs. 282 mg/dL) and cholesterol (97.1 vs. 119 mg/dL) than male mice. APO10LA-mitigated steatosis in males was associated with reduced hepatic total cholesterol (18%) and activated sirtuin 1 signaling, which resulted in reduced fatty acids (FAs) and TG synthesis markers [stearoyl-coenzyme A (CoA) desaturase protein, 71%; acetyl-CoA carboxylase phosphorylation, 79%; AMP-activated protein kinase phosphorylation, 67%], and elevated cholesterol efflux genes (cytochrome P450 family 7A1, 65%; ATP-binding cassette transporter G5/8, 11%). These APO10LA-mediated effects were not mimicked by lycopene supplementation. Intriguingly, steatosis inhibition by lycopene induced peroxisome proliferator-activated receptor (PPAR)α- and PPARγ-related genes in mesenteric adipose tissue (MAT) that increases mitochondrial uncoupling [cell death-inducing DNA fragmentation factor, α subunit-like effector a, 55%; PR domain-containing 16, 47%; uncoupling protein 3 (Ucp3), 55%], FA b-oxidation (PPARα, 53%; very long chain acyl-CoA dehydrogenase, 38%), and uptake (FA transport protein 4, 29%; lipoprotein lipase 43%). Expressions of 10 MAT PPAR-related genes were inversely correlated with steatosis score, suggesting that lycopene reduced steatosis by increasing MAT FA utilization. Conclusions: Our data suggest that lycopene and APO10LA inhibit HSFD-induced steatosis in BCO2-KO male mice through differential mechanisms. Sex disparity of BCO2-KO mice was observed in the outcomes of HSFD-induced liver steatosis and plasma lipids. [ABSTRACT FROM AUTHOR]

A cycle of cis-to-trans isomerization of the chromophore is intrinsic to vertebrate vision where rod and cone photoreceptors mediate dim- and bright-light vision, respectively. Daylight illumination can greatly exceed the rate at which the photoproduct can be recycled back to the chromophore by the canonical visual cycle. Thus, an additional supply pathway(s) must exist to sustain cone-dependent vision. Two-photon microscopy revealed that the eyes of the zebrafish (Danio rerio) contain high levels of 11-cis-retinyl esters (11-REs) within the retinal pigment epithelium. HPLC analyses demonstrate that 11-REs are bleached by bright light and regenerated in the dark. Pharmacologic treatment with all-trans-retinylamine (Ret-NH2), a potent and specific inhibitor of the trans-to-cis reisomerization reaction of the canonical visual cycle, impeded the regeneration of 11-REs. Intervention with 11-cis-retinol restored the regeneration of 11-REs in the presence of all-trans-Ret-NH2. We used the XOPS:mCFP transgenic zebrafish line with a functional cone-only retina to directly demonstrate that this 11-RE cycle is critical to maintain vision under bright-light conditions. Thus, our analyses reveal that a dark-generated pool of 11-REs helps to supply photoreceptors with the chromophore under the varying light conditions present in natural environments. [ABSTRACT FROM AUTHOR]

The critical role of retinoids (vitamin A and its derivatives) for vision, reproduction, and survival has been well established. Vitamin A is produced front dietary carotenoids such as β-carotene by centric cleavage via the enzyme BCO1. The biochemical and molecular identification of a second structurally related P-carotene metabolizing enzyme, BCO2, has led to a prolonged debate about its relevance in vitamin A biology. While BCO1 cleaves provitamin A carotenoids, BCO2 is more promiscuous and also metabolizes non-provitamin A carotenoids such as zeaxanthin into long-chain apo-carotenoids. Herein we demonstrate, in cell lines, that human BCO2 is associated with the inner mitochondrial membrane. Different human BCO2 isoforms possess cleavable N-terminal leader sequences critical for mitochondrial import. Subfractionation of murine hepatic mitochondria confirmed the localization of BCO2 to the inner mitochondrial membrane. Studies in BCO2-knockout mice revealed that zeaxanthin accumulates in the inner mitochondrial membrane; in contrast, β-carotene is retained predominantly in the cytoplasm. Thus, we provide evidence for a compartmentalization of carotenoid metabolism that prevents competition between BCO1 and BCO2 for the provitamin and the production of noncanonical β-carotene metabolites. [ABSTRACT FROM AUTHOR]

The demosponge Suberites domuncula has been described to contain high levels of a proteinaceous toxin, Suberitine, that displays haemolytic activityIn the present study this 7-8 kDa polypeptide has been isolated and was shown to exhibit also cytotoxic effects on cells of the same species. Addition of retinal, a recently identified metabolite of β-carotene that is abundantly present in S. domuncula was found to reduce both the haemolytic and the cell toxic activity of Suberitine at a molar ratio of 1:1. Spectroscopic analyses revealed that the interaction between β-carotene and Suberitine can be ascribed to a reversible energy transfer reaction. The enzyme that synthesises retinal in the sponge system is the β,β-carotene-15,15'-dioxygenase [carotene dioxygenase]. In order to clarify if this enzyme is the only β-carotene-metabolizing enzyme a further oxygenase had been identified and cloned, the (related) carotenoid oxygenase. In contrast to the dioxygenase, the carotenoid oxygenase could not degrade β-carotene or lycopene in Escherichia coli strains that produced these two carotenoids; therefore it had been termed related-carotenoid oxygenase. Exposure of primmorphs to light of different wavelengths from the visible spectrum resulted after 3 days in a strong upregulation of the dioxygenase in those 3D-cell aggregates that had been incubated with β-carotene. The strongest effect is seen with blue light at a maximum around 490 nm. It is concluded that the toxin Suberitine is non-covalently modified by retinal, the cleavage product from β-carotene via the enzyme carotene dioxygenase, a light inducible oxygenase. Hence, this study highlights that in S. domuncula the bioactive metabolite, retinal, has the property to detoxify its homologous toxin. [ABSTRACT FROM AUTHOR]

Carotenoids are the precursors for vitamin A and are proposed to prevent oxidative damage to cells. Mammalian genomes encode a family of structurally related nonheme iron oxygenases that modify double bonds of these compounds by oxidative cleavage and cis-to-trans isomerization. The roles of the family members BCMO1 and RPE65 for vitamin A production and vision have been well established. Surprisingly, we found that the third family member, β,β-carotene-9',10'-oxygenase (BCDO2), is a mitochondrial carotenoid-oxygenase with broad substrate specificity. In BCDO2-deficient mice, carotenoid homeostasis was abrogated, and carotenoids accumulated in several tissues. In hepatic mitochondria, accumulated carotenoids induced key markers of mitochondrial dysfunction, such as manganese superoxide dismutase (9-fold), and reduced rates of ADP-dependent respiration by 30%. This impairment was associated with an 8- to 9-fold induction of phosphor-MAP kinase and phosphor-AKT, markers of cell signaling pathways related to oxidative stress and disease. Administration of carotenoids to human HepG2 cells depolarized mitochondrial membranes and resulted in the production of reactive oxygen species. Thus, our studies in BCDO2-deficient mice and human cell cultures indicate that carotenoids can impair respiration and induce oxidative stress. Mammalian cells thus express a mitochondrial carotenoid-oxygenase that degrades carotenoids to protect these vital organelles. [ABSTRACT FROM AUTHOR]

Beta- carotene 15,15′- monooxygenase 1 knockout ( Bcmo1−/−) mice accumulate beta-carotene (BC) similarly to humans, whereas wild-type ( Bcmo1+/+) mice efficiently cleave BC. Bcmo1− /− mice are therefore suitable to investigate BC-induced alterations in gene expression in lung, assessed by microarray analysis. Bcmo1− /− mice receiving control diet had increased expression of inflammatory genes as compared to BC-supplemented Bcmo1− /− mice and Bcmo1+/+ mice that received either control or BC-supplemented diets. Differential gene expression in Bcmo1− /− mice was confirmed by real-time quantitative PCR. Histochemical analysis indeed showed an increase in inflammatory cells in lungs of control Bcmo1− /− mice. Supported by metabolite and gene-expression data, we hypothesize that the increased inflammatory response is due to an altered BC metabolism, resulting in an increased vitamin A requirement in Bcmo1−/− mice. This suggests that effects of BC may depend on inter-individual variations in BC-metabolizing enzymes, such as the frequently occurring human polymorphisms in BCMO1. [ABSTRACT FROM AUTHOR]

The enzyme retinol saturase (RetSat) catalyzes the saturation of all-trans-retinol to produce (R)-all-trans-13,14-dihydroretinol. As a peroxisome proliferator-activated receptor (PPAR) γ target, RetSat was shown to be required for adipocyte differentiation in the 3T3-L1 cell culture model. To understand the mechanism involved in this putative proadipogenic effect of RetSat, we studied the consequences of ablating RetSat expression on retinoid metabolism and adipose tissue differentiation in RetSat-null mice. Here, we report that RetSat-null mice have normal levels of retinol and retinyl palmitate in liver, serum, and adipose tissue, but, in contrast to wild-type mice, are deficient in the production of all-trans-13,14-dihydroretinol from dietary vitamin A. Despite accumulating more fat, RetSat-null mice maintained on either low-fat or high-fat diets gain weight and have similar rates of food intake as age- and gender-matched wildtype control littermates. This increased adiposity of RetSat-null mice is associated with up-regulation of PPARγ, a key transcriptional regulator of adipogenesis, and also its downstream target, fatty acid-binding protein 4 (FABP4/aP2). On the basis of these results, we propose that dihydroretinoids produced by RetSat control physiological processes that influence PPARγ activity and regulate lipid accumulation in mice. [ABSTRACT FROM AUTHOR]

In vertebrates, the absorption of a photon results in an 11-cis to all-trans isomerization of the retinylidene chromophore of cone and rod visual pigments. To sustain vision, metabolic pathways (visual cycles) have evolved that recycle all-trans-retinal back to 11-cis-retinal. The canonical visual cycle takes place in photoreceptor cells and the adjacent retinal pigment epithelium (RPE). Biochemical analyses provided evidence for the existence of an additional cone-specific visual cycle involving Müller glia cells, but none of its molecular components has yet been identified. Here we took advantage of the zebrafish retina to investigate the role of the cellular retinaldehydebinding protein CRALBP in this process. We found that the zebrafish genome encodes two cralbp paralogs: cralbp a and cralbp b. These paralogs are differentially expressed in the retina. Cralbp a is exclusively expressed in the RPE, and Cralbp b is localized to Müller cells. We used an antisense morpholino approach to knock down each cralbp paralog. Analysis of 11-cis-retinal levels revealed that visual chromophore regeneration is diminished under both conditions. Visual performance, as assessed by electroretinography, revealed reduced light sensitivity in both Cralbp a- and Cralbp b-deficient larvae, but it was more pronounced in Cralbp b-deficient larvae. Cralbp b-deficient larvae also exhibited significant deficits in their visual behavior. Together, these data demonstrate that Cralbp expression in Müller cells is essential for cone vision, thereby providing evidence that both the canonical and the alternative visual cycle depend on the same type of retinoid-binding protein. [ABSTRACT FROM AUTHOR]

Analyzes the blind Drosophila mutant fly to identify the key enzyme for the formation of vitamin A. Presence of mutations in the gene encoding; Contribution to a defect in vitamin A formation; Loss of the ability of the flies to form the visual chromophore.

Malaria, a devastating parasitic infection, is the leading cause of death in many developing countries. Unfortunately, the most deadliest causative agent of malaria, Plasmodium falciparum, has developed resistance to nearly all currently available antimalarial drugs. The P. falciparum Niemann-Pick type C1-related (PfNCR1) transporter has been identified as a druggable target, but its structure and detailed molecular mechanism are not yet available. Here, we present three structures of PfNCR1 with and without the functional inhibitor MMV009108 at resolutions between 2.98 and 3.81 Å using single-particle cryo-electron microscopy (cryo-EM), suggesting that PfNCR1 binds cholesterol and forms a cholesterol transport tunnel to modulate the composition of the parasite plasma membrane. Cholesterol efflux assays show that PfNCR1 is an exporter capable of extruding cholesterol from the membrane. Additionally, the inhibition mechanism of MMV009108 appears to be due to a direct blockage of PfNCR1, preventing this transporter from shuttling cholesterol. [ABSTRACT FROM AUTHOR]

Investigates the opine activation of the genes in the nopaline catabolic (noc) and octopine catabolic (occ) regions of Ti plasmids of Agrobacterium tumefaciens. Transcription start sites; Resection analysis; Similarities of the binding sites.

The precursor proteins of the carotenogenic enzymes geranylgeranyl diphosphate synthase, phytoene synthase, phytoene desaturase and lycopene cyclase were imported into isolated pea chloroplasts. Geranylgeranyl diphosphate synthase remained soluble in the stroma in a free form and phytoene synthase associated to thylakoid membranes upon import, both as expected. Surprisingly, phytoene desaturase and lycopene cyclase, which strongly depend on membrane association for enzymatic activity, also remained soluble in the chloroplast stroma. The soluble forms of these enzymes were, however, still competent for membrane-association, e.g. with protein-free liposomal membranes. Indeed the soluble forms of phytoene synthase, phytoene desaturase and lycopene cyclase occurred as ATP- and cold-sensitive high-molecularmass complexes. Gel-filtration experiments and blue native-PAGE plus autoradiography and western blot analysis indicated a participation of the chloroplast 60-kDa chaperonin (Cpn60) in the soluble highmolecular-mass complexes of imported carotenogenic enzymes. Finally, it was inferred that a membranebound regulatory factor plays a decisive role in membrane-binding. [ABSTRACT FROM AUTHOR]

The photochemistry of vision employs opsins and geometric isomerization of their covalently bound retinylidine chromophores. In different animal classes, these light receptors associate with distinct G proteins that either hyperpolarize or depolarize photoreceptor membranes. Vertebrates also use the acidic form of chromophore, retinoic acid, as the ligand of nuclear hormone receptors that orchestrate eye development. To establish and sustain these processes, animals must acquire carotenoids from the diet, transport them, and metabolize them to chromophore and retinoic acid. The understanding of carotenoid metabolism, however, lagged behind our knowledge about the biology of their receptor molecules. In the past decades, much progress has been made in identifying the genes encoding proteins that mediate the transport and enzymatic transformations of carotenoids and their retinoid metabolites. Comparative analysis in different animal classes revealed how evolutionary tinkering with a limited number of genes evolved different biochemical strategies to supply photoreceptors with chromophore. Mutations in these genes impair carotenoid metabolism and induce various ocular pathologies. This review summarizes this advancement and introduces the involved proteins, including the homeostatic regulation of their activities. [ABSTRACT FROM AUTHOR]

Carotenoids exert a rich variety of physiological functions in mammals and are beneficial for human health. These lipids are acquired from the diet and metabolized to apocarotenoids, including retinoids (vitamin A and its metabolites). The small intestine is a major site for their absorption and bioconversion. From here, carotenoids and their metabolites are distributed within the body in triacylglycerol-rich lipoproteins to support retinoid signaling in peripheral tissues and photoreceptor function in the eyes. In recent years, much progress has been made in identifying carotenoid metabolizing enzymes, transporters, and binding proteins. A diet-responsive regulatory network controls the activity of these components and adapts carotenoid absorption and bioconversion to the bodily requirements of these lipids. Genetic variability in the genes encoding these components alters carotenoid homeostasis and is associated with pathologies. We here summarize the advanced state of knowledge about intestinal carotenoid metabolism and its impact on carotenoid and retinoid homeostasis of other organ systems, including the eyes, liver, and immune system. The implication of the findings for science-based intake recommendations for these essential dietary lipids is discussed. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro. Unlabelled Image • Factors determining carotenoid bio-accessibility and absorption. • Biochemical properties of major carotenoid and retinoid metabolizing enzymes. • A diet-responsive regulatory network controls intestinal carotenoid absorption and bioconversion to retinoids. • Crosstalk between dietary carotenoids and gastrointestinal immunity. • Diet and genetics affect carotenoid absorption and plasma response in humans. [ABSTRACT FROM AUTHOR]

Background: Vitamin A (VA) deficiency is a public health problem in some countries. Fortification, supplementation, and increased provitamin A consumption through biofortification are efficacious, but monitoring is needed due to risk of excessive VA intake when interventions overlap.Objectives: Two studies in 28-36-d-old male Mongolian gerbils simulated exposure to multiple VA interventions to determine the effects of provitamin A carotenoid consumption from biofortified maize and carrots and preformed VA fortificant on status.Methods: Study 1 was a 2 × 2 × 2 factorial design (n = 85) with high-β-carotene maize, orange carrots, and VA fortification at 50% estimated gerbil needs, compared with white maize and white carrot controls. Study 2 was a 2 × 3 factorial design (n = 66) evaluating orange carrot and VA consumption through fortification at 100% and 200% estimated needs. Both studies utilized 2-wk VA depletion, baseline evaluation, 9-wk treatments, and liver VA stores by HPLC. Intestinal scavenger receptor class B member 1 (Scarb1), β-carotene 15,15'-dioxygenase (Bco1), β-carotene 9',10'-oxygenase (Bco2), intestine-specific homeobox (Isx), and cytochrome P450 26A1 isoform α1 (Cyp26a1) expression was analyzed by qRT-PCR in study 2.Results: In study 1, liver VA concentrations were significantly higher in orange carrot (0.69 ± 0.12 μmol/g) and orange maize groups (0.52 ± 0.21 μmol/g) compared with baseline (0.23 ± 0.069 μmol/g) and controls. Liver VA concentrations from VA fortificant alone (0.11 ± 0.053 μmol/g) did not differ from negative control. In study 2, orange carrot significantly enhanced liver VA concentrations (0.85 ± 0.24 μmol/g) relative to baseline (0.43 ± 0.14 μmol/g), but VA fortificant alone (0.42 ± 0.21 μmol/g) did not. Intestinal Scarb1 and Bco1 were negatively correlated with increasing liver VA concentrations (P < 0.01, r2 = 0.25-0.27). Serum retinol concentrations did not differ.Conclusions: Biofortified carrots and maize without fortification prevented VA deficiency in gerbils. During adequate provitamin A dietary intake, preformed VA intake resulted in excessive liver stores in gerbils, despite downregulation of carotenoid absorption and cleavage gene expression. [ABSTRACT FROM AUTHOR]

Background: Astaxanthin is a red lipophilic carotenoid that is often undetectable in human plasma due to the limited supply in typical Western diets. Despite its presence at lower than detectable concentrations, previous clinical feeding studies have reported that astaxanthin exhibits potent antioxidant properties.Objective: We examined astaxanthin accumulation and its effects on gut microbiota, inflammation, and whole-body metabolic homeostasis in wild-type C57BL/6 J (WT) and β-carotene oxygenase 2 (BCO2) knockout (KO) mice.Methods: Six-wk-old male and female BCO2 KO and WT mice were provided with either nonpurified AIN93M (e.g., control diet) or the control diet supplemented with 0.04% astaxanthin (wt/wt) ad libitum for 8 wk. Whole-body energy expenditure was measured by indirect calorimetry. Feces were collected from individual mice for short-chain fatty acid assessment. Hepatic astaxanthin concentrations and liver metabolic markers, cecal gut microbiota profiling, inflammation markers in colonic lamina propria, and plasma samples were assessed. Data were analyzed by 3-way ANOVA followed by Tukey's post hoc analysis.Results: BCO2 KO but not WT mice fed astaxanthin had ∼10-fold more of this compound in liver than controls (P < 0.05). In terms of the microbiota composition, deletion of BCO2 was associated with a significantly increased abundance of Mucispirillum schaedleri in mice regardless of gender. In addition to more liver astaxanthin in male KO compared with WT mice fed astaxanthin, the abundance of gut Akkermansia muciniphila was 385% greater, plasma glucagon-like peptide 1 was 27% greater, plasma glucagon and IL-1β were 53% and 30% lower, respectively, and colon NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation was 23% lower (all P < 0.05) in male KO mice than the WT mice.Conclusions: Astaxanthin affects the gut microbiota composition in both genders, but the association with reductions in local and systemic inflammation, oxidative stress, and improvement of metabolic homeostasis only occurs in male mice. [ABSTRACT FROM AUTHOR]

A delicate balance between photon absorption for vision and the protection of photoreceptors from light damage is pivotal for ocular health. This equilibrium is governed by the lightabsorbing 11-cis-retinylidene chromophore of visual pigments, which, upon bleaching, transforms into all-trans-retinal and undergoes regeneration through an enzymatic pathway, named the visual cycle. Chemical side reactions of retinaldehyde during the recycling process can generate by-products that may result in a depletion of retinoids. In our study, we have clarified the crucial roles played by melanin pigmentation and the retinoid transporter STRA6 in preventing this loss and preserving the integrity of the visual cycle. Our experiments initially confirmed that consecutive green and blue light bleaching of isolated bovine rhodopsin produced 9-cis and 13-cis retinal. The same unusual retinoids were found in the retinas of mice exposed to intense light, with elevated concentrations observed in albino mice. Examining the metabolic fate of these visual cycle byproducts revealed that 9-cis-retinal, but not 13-cis-retinal, was recycled back to all-trans-retinal through an intermediate called isorhodopsin. However, investigations in Stra6 knockout mice unveiled that the generation of these visual cycle byproducts correlated with a light-induced loss of ocular retinoids and visual impairment. Collectively, our findings uncover important novel aspects of visual cycle dynamics, with implications for ocular health and photoreceptor integrity. [ABSTRACT FROM AUTHOR]

Various species of ascomycete fungi synthesize the carboxylic carotenoid neurosporaxanthin. The unique chemical structure of this xanthophyll reveals that: (1) Its carboxylic end and shorter length increase the polarity of neurosporaxanthin in comparison to other carotenoids, and (2) it contains an unsubstituted β-ionone ring, conferring the potential to form vitamin A. Previously, neurosporaxanthin production was optimized in Fusarium fujikuroi, which allowed us to characterize its antioxidant properties in in vitro assays. In this study, we assessed the bioavailability of neurosporaxanthin compared to other provitamin A carotenoids in mice and examined whether it can be cleaved by the two carotenoid-cleaving enzymes: β-carotene-oxygenase 1 (BCO1) and 2 (BCO2). Using Bco1−/−Bco2−/− mice, we report that neurosporaxanthin displays greater bioavailability than β-carotene and β-cryptoxanthin, as evidenced by higher accumulation and decreased fecal elimination. Enzymatic assays with purified BCO1 and BCO2, together with feeding studies in wild-type, Bco1−/−, Bco2−/−, and Bco1−/−Bco2−/− mice, revealed that neurosporaxanthin is a substrate for either carotenoid-cleaving enzyme. Wild-type mice fed neurosporaxanthin displayed comparable amounts of vitamin A to those fed β-carotene. Together, our study unveils neurosporaxanthin as a highly bioavailable fungal carotenoid with provitamin A activity, highlighting its potential as a novel food additive. The fungal carboxylic carotenoid neurosporaxanthin is absorbed at greater rates than other carotenoids, and it can be cleaved to form vitamin A in mice. These findings suggest neurosporaxanthin could be an attractive vitamin A source for humans. [ABSTRACT FROM AUTHOR]

A footnote for the article "Conversion of ß-Carotene to Retinal Pigment," by Hans K. Biesalski and colleagues, which mentions ways to combat vitamin A deficiency is presented.

Vitamin A is an essential nutrient crucial to ensuring proper mammalian embryonic development. β-Carotene is the most prevalent form of vitamin A in food that, when transferred in its intact form from mother to the developing tissues, can serve as an in situ source of retinoic acid, the active form of vitamin A. We have previously provided evidence that the maternal-fetal transfer of β-carotene across the placenta is mediated by lipoproteins and that β-carotene itself regulates placenta lipoprotein biogenesis by means of its derivatives β-apo-10′-carotenoids and retinoic acid. These metabolites exert antagonistic transcriptional activity on placental microsomal triglyceride transfer protein (MTP) and apolipoprotein B (APOB), two key players of lipoprotein biosynthesis. Here, we analyzed the time-dependency of this regulation over the course of 24 h upon a single maternal administration of β-carotene. We also tested the hypothesis that the transcriptional repressor intestine-specific homeobox (ISX) plays a role in the regulation of Mttp in placenta. We observed that ISX is expressed in placenta of mouse dams and is regulated by β-carotene availability. Furthermore, we demonstrated that the absence of Isx disrupts the β-carotene-mediated regulation of placental MTP. We also showed that this mechanism is organ-specific, as it was not observed in enterocytes of the intestine, a major place of Isx expression. Therefore, we identified ISX as a "master" regulator of a placental β-carotene-dependent transcriptional regulatory cascade that fine-tunes the flux of provitamin A carotenoid towards the developing fetus. • β-Carotene metabolites regulate placental MTP is a time-dependent manner. • The intestine-specific homeobox (ISX) transcription factor is expressed in placenta. • β-Carotene regulation of placental MTP is dependent on ISX. • β-Carotene regulation of MTP is organ-specific. [ABSTRACT FROM AUTHOR]

Some mammalian tissues uniquely concentrate carotenoids, but the underlying biochemical mechanism for this accumulation has not been fully elucidated. For instance, the central retina of the primate eyes displays high levels of the carotenoids, lutein, and zeaxanthin, whereas the pigments are largely absent in rodent retinas. We previously identified the scavenger receptor class B type 1 and the enzyme ß-carotene-oxygenase-2 (BCO2) as key components that determine carotenoid concentration in tissues. We now provide evidence that Aster (GRAM-domain-containing) proteins, recently recognized for their role in nonvesicular cholesterol transport, engage in carotenoid metabolism. Our analyses revealed that the StART-like lipid binding domain of Aster proteins can accommodate the bulky pigments and bind them with high affinity. We further showed that carotenoids and cholesterol compete for the same binding site. We established a bacterial test system to demonstrate that the StART-like domains of mouse and human Aster proteins can extract carotenoids from biological membranes. Mice deficient for the carotenoid catabolizing enzyme BCO2 concentrated carotenoids in Aster-B protein-expressing tissues such as the adrenal glands. Remarkably, Aster-B was expressed in the human but not in the mouse retina. Within the retina, Aster-B and BCO2 showed opposite expression patterns in central versus peripheral parts. Together, our study unravels the biochemical basis for intracellular carotenoid transport and implicates Aster-B in the pathway for macula pigment concentration in the human retina. [ABSTRACT FROM AUTHOR]

The article presents a study which explores genomic consequences of domestication of the Siamese fighting fish. It demonstrate how simple genetic architectures paired with anatomical modularity can lead to vast phenotypic diversity generated during animal domestication and launch betta as a powerful new system for evolutionary genetics.

The enzyme b-carotene oxygenase 2 (BCO2) converts carotenoids into more polar metabolites. Studies in mammals, fish, and birds revealed that BCO2 controls carotenoid homeostasis and is involved in the pathway for vitamin A production. However, it is controversial whether BCO2 function is conserved in humans, because of a 4-amino acid long insertion caused by a splice acceptor site polymorphism. We here show that human BCO2 splice variants, BCO2a and BCO2b, are expressed as pre-proteins with mitochondrial targeting sequence (MTS). The MTS of BCO2a directed a green fluorescent reporter protein to the mitochondria when expressed in ARPE-19 cells. Removal of the MTS increased solubility of BCO2a when expressed in Escherichia coli and rendered the recombinant protein enzymatically active. The expression of the enzymatically active recombinant human BCO2a was further improved by codon optimization and its fusion with maltose-binding protein. Introduction of the 4-amino acid insertion into mouse Bco2 did not impede the chimeric enzyme's catalytic proficiency. We further showed that the chimeric BCO2 displayed broad substrate specificity and converted carotenoids into two ionones and a central C14-apocarotendial by oxidative cleavage reactions at C9,C10 and C9',C10'. Thus, our study demonstrates that human BCO2 is a catalytically competent enzyme. Consequently, information on BCO2 becomes broadly applicable in human biology with important implications for the physiology of the eyes and other tissues. [ABSTRACT FROM AUTHOR]

Apocarotenoids are important signaling molecules generated from carotenoids through the action of carotenoid cleavage dioxygenases (CCDs). These enzymes have a remarkable ability to cleave carotenoids at specific alkene bonds while leaving chemically similar sites within the polyene intact. Although several bacterial and eukaryotic CCDs have been characterized, the long-standing goal of experimentally visualizing a CCD–carotenoid complex at high resolution to explain this exquisite regioselectivity remains unfulfilled. CCD genes are also present in some archaeal genomes, but the encoded enzymes remain uninvestigated. Here, we address this knowledge gap through analysis of a metazoan-like archaeal CCD from Candidatus Nitrosotalea devanaterra (NdCCD). NdCCD was active toward β-apocarotenoids but did not cleave bicyclic carotenoids. It exhibited an unusual regiospecificity, cleaving apocarotenoids solely at the C14′–C13′ alkene bond to produce β-apo-14′-carotenals. The structure of NdCCD revealed a tapered active site cavity markedly different from the broad active site observed for the retinal-forming Synechocystis apocarotenoid oxygenase (SynACO) but similar to the vertebrate retinoid isomerase RPE65. The structure of NdCCD in complex with its apocarotenoid product demonstrated that the site of cleavage is defined by interactions along the substrate binding cleft as well as selective stabilization of reaction intermediates at the scissile alkene. These data on the molecular basis of CCD catalysis shed light on the origins of the varied catalytic activities found in metazoan CCDs, opening the possibility of modifying their activity through rational chemical or genetic approaches. [ABSTRACT FROM AUTHOR]

Carotenoid cleavage dioxygenases (CCDs) use a nonheme Fe(II) cofactor to split alkene bonds of carotenoid and stilbenoid substrates. The iron centers of CCDs are typically five-coordinate in their resting states, with solvent occupying an exchangeable site. The involvement of this iron-bound solvent in CCD catalysis has not been experimentally addressed, but computational studies suggest two possible roles. 1) Solvent dissociation provides a coordination site forO2, or 2) solvent remains bound to iron but changes its equilibrium position to allow O2 binding and potentially acts as a proton source. To test these predictions, we investigated isotope effects (H2O versus D2O) on two stilbenoidcleaving CCDs, Novosphingobium aromaticivorans oxygenase 2 (NOV2) and Neurospora crassa carotenoid oxygenase 1 (CAO1), using piceatannol as a substrate. NOV2 exhibited an inverse isotope effect (kH/kD~0.6) in an air-saturated buffer, suggesting that solvent dissociates from iron during the catalytic cycle. By contrast, CAO1 displayed a normal isotope effect (kH/kD ~ 1.7), suggesting proton transfer in the rate-limiting step. X-ray absorption spectroscopyonNOV2andCAO1indicated that the protonation states of the iron ligands are unchanged within pH 6.5-8.5 and that the Fe(II)-aquo bond is minimally altered by substrate binding. We pinpointed the origin of the differential kinetic behaviors ofNOV2 and CAO1 to a single amino acid difference near the solvent-binding site of iron, and X-ray crystallography revealed that the substitution alters binding of diffusible ligands to the iron center. We conclude that solvent-iron dissociation and proton transfer are both associated with the CCD catalytic mechanism. [ABSTRACT FROM AUTHOR]

Carotenoids can be enzymatically converted to apocarotenoids by carotenoid cleavage dioxygenases. Insect genomes encode only one member of this ancestral enzyme family. We cloned and characterized the ninaB genes from the silk worm (Bombyx mori) and the flour beetle (Tribolium castaneum). We expressed BmNinaB and TcNinaB in E. coli and analyzed their biochemical properties. Both enzymes catalyzed a conversion of carotenoids into cis -retinoids. The enzymes catalyzed a combined trans to cis isomerization at the C11, C12 double bond and oxidative cleavage reaction at the C15, C15′ bond of the carotenoid carbon backbone. Analyses of the spatial and temporal expression patterns revealed that ninaB genes were differentially expressed during the beetle and moth life cycles with high expression in reproductive organs. In Bombyx mori, ninaB was almost exclusively expressed in female reproductive organs of the pupa and adult. In Tribolium castaneum , low expression was found in reproductive organs of females but high expressions in male reproductive organs of the pupa and imagoes. We performed RNAi experiments to characterize the role of NinaB in insect reproduction. We observed that RNAi treatment significantly decreased the expression levels of BmninaB and TcninaB and reduced the egg laying capacity of both insects. Together, our study revealed that NinaB's unique enzymatic properties are well conserved among insects and implicate NinaB function in insect reproduction. Image 1 • NinaB's oxygenase and isomerase activity for the bicyclic carotenoid substrates is well conserved among insects. • Both BmninaB and TcninaB are differentially expressed between male and female reproductive organs. • RNAi of BmninaB and TcninaB reduce the capacity of reproduction in both Bombyx mori and Tribolium castaneum. [ABSTRACT FROM AUTHOR]

Carotenoid cleavage oxygenases (CCO) are non-heme iron enzymes that catalyze oxidative cleavage of alkene bonds in carotenoid and stilbenoid substrates. Previously, we showed that the iron cofactor of CAO1, a resveratrol-cleaving member of this family, can be substituted with cobalt to yield a catalytically inert enzyme useful for trapping active site-bound stilbenoid substrates for structural characterization. Metal substitution may provide a general method for identifying the natural substrates for CCOs in addition to facilitating structural and biophysical characterization of CCO-carotenoid complexes under normal aerobic conditions. Here, we demonstrate the general applicability of cobalt substitution in a prototypical carotenoid cleaving CCO, apocarotenoid oxygenase (ACO) from Synechocystis. Among the non-native divalent metals investigated, cobalt was uniquely able to stably occupy the ACO metal binding site and inhibit catalysis. Analysis by X-ray crystallography and X-ray absorption spectroscopy demonstrate that the Co(II) forms of both ACO and CAO1 exhibit a close structural correspondence to the native Fe(II) enzyme forms. Hence, cobalt substitution is an effective strategy for generating catalytically inert but structurally intact forms of CCOs. [ABSTRACT FROM AUTHOR]

The intestinal epithelium is a major site for the conversion of dietary β-carotene to retinaldehyde by the enzyme BCO1. The majority of retinaldehyde is further metabolized to retinol (vitamin A), esterified and packaged into triacylglycerol-rich chylomicrons for bodily distribution. Some serve on-site for the synthesis of retinoic acid, a hormone-like compound, which exerts pleiotropic and dominant effects on gastrointestinal immunity. We report here that the intestine-specific homeobox protein ISX is critical to control themetabolic flow of β-carotene through this important branching point of vitamin A metabolism. This transcription factor represses Bco1 gene expression in response to retinoic acid signaling. In ISX-deficient mice, uncontrolled Bco1 gene expression led to increased retinoid production in the intestine. Systemically, this production resulted in highly elevated hepatic retinoid stores. In the intestine, it increased the expression of retinoic acid-inducible target genes such as Aldh1a2, Dhrs3, and Ccr9. The β-carotene-inducible disruption of retinoid homeostasis affected gut-homing and differentiation of lymphocytes and displayed morphologically in large lymphoid follicles along the intestine. Furthermore, it was associated with an infiltration of the pancreas by gut-derived lymphocytes that manifested as a pancreatic insulitis with β-islet cell destruction and systemic glucose intolerance. Thus, our study identifies an important molecular interlink between diet and immunity and indicates that vitamin A homeostasis must be tightly controlled by ISX to maintain immunity and tolerance at the intestinal barrier. [ABSTRACT FROM AUTHOR]

Carotenoid cleavage oxygenases (CCOs) are non-heme, Fe(II)-dependent enzymes that participate in biologically important metabolic pathways involving carotenoids and apocarotenoids, including retinoids, stilbenes, and related compounds. CCOs typically catalyze the cleavage of non-aromatic double bonds by dioxygen (O2) to form aldehyde or ketone products. Expressed only in vertebrates, the RPE65 sub-group of CCOs catalyzes a non-canonical reaction consisting of concerted ester cleavage and trans-cis isomerization of all-trans-retinyl esters. It remains unclear whether the former group of CCOs functions as mono- or di-oxygenases. Additionally, a potential role for O2 in catalysis by the RPE65 group of CCOs has not been evaluated to date. Here, we investigated the pattern of oxygen incorporation into apocarotenoid products of Synechocystis apocarotenoid oxygenase. Reactions performed in the presence of 18O-labeled water and 18O2 revealed an unambiguous dioxygenase pattern of O2 incorporation into the reaction products. Substitution of Ala for Thr at position 136 of apocarotenoid oxygenase, a site predicted to govern the mono-versus dioxygenase tendency of CCOs, greatly reduced enzymatic activity without altering the dioxygenase labeling pattern. Reevaluation of the oxygen-labeling pattern of the resveratrol-cleaving CCO, NOV2, previously reported to be a monooxygenase, using a purified enzyme sample revealed that it too is a dioxygenase. We also demonstrated that bovine RPE65 is not dependent on O2 for its cleavage/isomerase activity. In conjunction with prior research, the results of this study resolve key issues regarding the utilization of O2 by CCOs and indicate that dioxygenase activity is a feature common among double bondcleaving CCOs. [ABSTRACT FROM AUTHOR]

A family of enzymes collectively referred to as carotenoid cleavage oxygenases is responsible for oxidative conversion of carotenoids into apocarotenoids, including retinoids (vitaminA and its derivatives). A member of this family, the β-carotene 9,10-dioxygenase (BCO2), converts xanthophylls to rosafluene and ionones. Animals deficient in BCO2 highlight the critical role of the enzyme in carotenoid clearance as accumulation of these compounds occur in tissues. Inactivation of the enzyme by a four-amino acid-long insertion has recently been proposed to underlie xanthophyll concentration in the macula of the primate retina. Here, we focused on comparing the properties of primate and murine BCO2s. We demonstrate that the enzymes display a conserved structural fold and subcellular localization. Low temperature expression and detergent choice significantly affected binding and turnover rates of the recombinant enzymes with various xanthophyll substrates, including the unique macula pigment meso-zeaxanthin. Mice with genetically disrupted carotenoid cleavage oxygenases displayed adipose tissue rather than eye-specific accumulation of supplemented carotenoids. Studies in a human hepatic cell line revealed that BCO2 is expressed as an oxidative stress-induced gene. Our studies provide evidence that the enzymatic function of BCO2 is conserved in primates and link regulation of BCO2 gene expression with oxidative stress that can be caused by excessive carotenoid supplementation. [ABSTRACT FROM AUTHOR]

Recent evidence suggests an important role for outer retinal cells in the pathogenesis of diabetic retinopathy (DR). Here we investigated the effect of the visual cycle inhibitor retinylamine (Ret-NH2) on the development of early DR lesions. Wild-type (WT) C57BL/6J mice (male, 2 months old when diabetes was induced) were made diabetic with streptozotocin, and some were given Ret-NH2 once per week. Lecithin-retinol acyltransferase (LRAT)-deficient mice and P23H mutant mice were similarly studied. Mice were euthanized after 2 (WT and Lrat-/-) and 8 months (WT) of study to assess vascular histopathology, accumulation of albumin, visual function, and biochemical and physiological abnormalities in the retina. Non-retinal effects of Ret-NH2 were examined in leukocytes treated in vivo. Superoxide generation and expression of inflammatory proteins were significantly increased in retinas of mice diabetic for 2 or 8 months, and the number of degenerate retinal capillaries and accumulation of albumin in neural retina were significantly increased in mice diabetic for 8 months compared with nondiabetic controls. Administration of Ret-NH2 once per week inhibited capillary degeneration and accumulation of albumin in the neural retina, significantly reducing diabetes-induced retinal superoxide and expression of inflammatory proteins. Superoxide generation also was suppressed in Lrat-/- diabetic mice. Leukocytes isolated from diabetic mice treated with Ret-NH2 caused significantly less cytotoxicity to retinal endothelial cells ex vivo than did leukocytes from control diabetics. Administration of Ret-NH2 once per week significantly inhibited the pathogenesis of lesions characteristic of earlyDRin diabetic mice. The visual cycle constitutes a novel target for inhibition of DR. [ABSTRACT FROM AUTHOR]

Carotenoid cleavage enzymes (CCEs) constitute a group of evolutionarily related proteins that metabolize a variety of carotenoid and non-carotenoid substrates. Typically, these enzymes utilize a non-heme iron center to oxidatively cleave a carbon-carbon double bond of a carotenoid substrate. Some members also isomerize specific double bonds in their substrates to yield cis-apocarotenoid products. The apocarotenoid oxygenase from Synechocystis has been hypothesized to represent one such member of this latter category of CCEs. Here, we developed a novel expression and purification protocol that enabled production of soluble, native ACO in quantities sufficient for high resolution structural and spectroscopic investigation of its catalytic mechanism. High performance liquid chromatography and Raman spectroscopy revealed that ACO exclusively formed all-trans products. We also found that linear polyoxyethylene detergents previously used for ACO crystallization strongly inhibited the apocarotenoid oxygenase activity of the enzyme. We crystallized the native enzyme in the absence of apocarotenoid substrate and found electron density in the active site that was similar in appearance to the density previously attributed to a di-cis-apocarotenoid intermediate. Our results clearly demonstrated that ACO is in fact a non-isomerizing member of the CCE family. These results indicate that careful selection of detergent is critical for the success of structural studies aimed at elucidating structures of CCE-carotenoid/retinoid complexes. [ABSTRACT FROM AUTHOR]

Mammalian genomes encode two provitamin A-converting enzymes as follows: the β-carotene-15,15'-oxygenase (BCO1) and the β-carotene-9',10'-oxygenase (BCO2). Symmetric cleavage by BCO1 yields retinoids (β-15'-apocarotenoids, C20), whereas eccentric cleavage by BCO2 produces long-chain (>C20) apocarotenoids. Here, we used genetic and biochemical approaches to clarify the contribution of these enzymes to provitamin A metabolism. We subjected wild type, Bco1-/-, Bco2-/-, and Bco1-/-Bco2-/- double knock-out mice to a controlled diet providing β-carotene as the sole source for apocarotenoid production. This study revealed that BCO1 is critical for retinoid homeostasis. Genetic disruption of BCO1 resulted in β-carotene accumulation and vitamin A deficiency accompanied by a BCO2-dependent production of minor amounts of β-apo-10'-carotenol (APO10ol). We found that APO10ol can be esterified and transported by the same proteins as vitamin A but with a lower affinity and slower reaction kinetics. In wild type mice, APO10ol was converted to retinoids by BCO1. We also show that a stepwise cleavage by BCO2 and BCO1 with APO10ol as an intermediate could provide a mechanism to tailor asymmetric carotenoids such as β-cryptoxanthin for vitamin A production. In conclusion, our study provides evidence that mammals employ both carotenoid oxygenases to synthesize retinoids from provitamin A carotenoids. [ABSTRACT FROM AUTHOR]

Animals perceiving light through visual pigments have evolved pathways for absorbing, transporting, and metabolizing the precursors essential for synthesis of their retinylidene chromophores. Over the past decades, our understanding of this metabolism has grown significantly. Through genetic manipulation, researchers gained insights into the metabolic complexity of the pathways mediating the flow of chromophore precursors throughout the body, and their enrichment within the eyes. This exploration has identified transport proteins and metabolizing enzymes for these essential lipids and has revealed some of the fundamental regulatory mechanisms governing this process. What emerges is a complex framework at play that maintains ocular retinoid homeostasis and functions. This review summarizes the recent advancements and highlights future research directions that may deepen our understanding of this complex metabolism., (Copyright © 2025. Published by Elsevier Inc.)