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A family of enzymes collectively referred to as carotenoid cleavage oxygenases is responsible for oxidative conversion of carotenoids into apocarotenoids, including retinoids (vitamin A 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.

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.

Carotenoids affect a rich variety of physiological functions in nature and are beneficial for human health. However, knowledge about their biological action and the consequences of their dietary accumulation in mammals is limited. Progress in this research field is limited by the expeditious metabolism of carotenoids in rodents and the confounding production of apocarotenoid signaling molecules. Herein, we established a mouse model lacking the enzymes responsible for carotenoid catabolism and apocarotenoid production, fed on either a β-carotene- or a zeaxanthin-enriched diet. Applying a genome wide microarray analysis, we assessed the effects of the parent carotenoids on the liver transcriptome. Our analysis documented changes in pathways for liver lipid metabolism and mitochondrial respiration. We biochemically defined these effects, and observed that β-carotene accumulation resulted in an elevation of liver triglycerides and liver cholesterol, while zeaxanthin accumulation increased serum cholesterol levels. We further show that carotenoids were predominantly transported within HDL particles in the serum of mice. Finally, we provide evidence that carotenoid accumulation influenced whole-body respiration and energy expenditure. Thus, we observed that accumulation of parent carotenoids interacts with lipid metabolism and that structurally related carotenoids display distinct biological functions in mammals.

Vitamin A bound to retinol binding protein 4 (RBP4) constitutes the major transport mode for retinoids in fasting circulation. Emerging evidence suggests that membrane protein, STRA6 (stimulated by retinoic acid 6), is the RBP4 receptor and vitamin A channel; however, the role of STRA6 in vitamin A homeostasis remains to be defined in vivo. We subjected Stra6-knockout mice to diets sufficient and insufficient for vitamin A and used heterozygous siblings as controls. We determined vitamin A levels of the eyes, brain, and testis, which highly express Stra6, as well as of tissues with low expression, such as lung and fat. We also studied the consequence of STRA6 deficiency on retinoid-dependent processes in tissues. Furthermore, we examined how STRA6 deficiency affected retinoid homeostasis of the aging mouse. The picture that emerged indicates a critical role for STRA6 in the transport of vitamin A across blood–tissue barriers in the eyes, brain, and testis. Concurrently, fat and lung rely on dietary vitamin A. In testis and brain, Stra6 expression was regulated by vitamin A. In controls, this regulation reduced vitamin A consumption when the dietary supply was limited, sequestering it for the eye. Thus, STRA6 is critical for vitamin A homeostasis and the adaption of this process to the fluctuating supply of the vitamin.—Kelly, M., Widjaja-Adhi, M. A. K., Palczewski, G., von Lintig, J. Transport of vitamin A across blood–tissue barriers is facilitated by STRA6.

Carotenoids are currently investigated regarding their potential to lower the risk of chronic disease and to combat vitamin A deficiency. Surprisingly, responses to dietary supplementation with these compounds are quite variable between individuals. Genome-wide studies have associated common genetic polymorphisms in the BCO1 gene with this variability. The BCO1 gene encodes an enzyme that is expressed in the intestine and converts provitamin A carotenoids to vitamin A-aldehyde. However, it is not clear how this enzyme can impact the bioavailability and metabolism of other carotenoids such as xanthophyll. We here provide evidence that BCO1 is a key component of a regulatory network that controls the absorption of carotenoids and fat-soluble vitamins. In this process, conversion of β-carotene to vitamin A by BCO1 induces via retinoid signaling the expression of the intestinal homeobox transcription factor ISX. Subsequently, ISX binds to conserved DNA-binding motifs upstream of the BCO1 and SCARB1 genes. SCARB1 encodes a membrane protein that facilitates absorption of fat-soluble vitamins and carotenoids. In keeping with its role as a transcriptional repressor, SCARB1 protein levels are significantly increased in the intestine of ISX-deficient mice. This increase results in augmented absorption and tissue accumulation of xanthophyll carotenoids and tocopherols. Our study shows that fat-soluble vitamin and carotenoid absorption is controlled by a BCO1-dependent negative feedback regulation. Thus, our findings provide a molecular framework for the controversial relationship between genetics and fat-soluble vitamin status in the human population.