Glenn P. Lobo

A mitochondrial enzyme degrades carotenoids and protects against oxidative stress

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.—Amengual, J., Lobo, G. P., Golczak, M., Li, H. N. M., Klimova, T., Hoppel, C. L., Wyss, A., Palczewski, K., von Lintig, J. A mitochondrial enzyme degrades carotenoids and protects against oxidative stress. FASEB J. 25, 948–959 (2011). www.fasebj.org

ISX is a retinoic acid-sensitive gatekeeper that controls intestinal β,β-carotene absorption and vitamin A production

The uptake of dietary lipids from the small intestine is a complex process that depends on the activities of specific membrane receptors with yet unknown regulatory mechanisms. Using both mouse models and human cell lines, we show here that intestinal lipid absorption by the scavenger receptor class B type 1 (SR‐BI) is subject to control by retinoid signaling. Retinoic acid via retinoic acid receptors induced expression of the intestinal transcription factor ISX. ISX then repressed the expression of SR‐B1 and the carotenoid‐15,15′‐oxygenase Bcmo1. BCMO1 acts downstream of SR‐BI and converts absorbed β,β‐carotene to the retinoic acid precursor, retinaldehyde. Using BCMO1‐knockout mice, we demonstrated increased intestinal SR‐BI expression and systemic β,β‐carotene accumulation. SR‐BI‐dependent accumulation of β,β‐carotene was prevented by dietary retinoids that induced ISX expression. Thus, our study revealed a diet‐responsive regulatory network that controls β,β‐carotene absorption and vitamin A production by negative feedback regulation. The role of SR‐BI in the intestinal absorption of other dietary lipids, including cholesterol, fatty acids, and tocopherols, implicates retinoid signaling in the regulation of lipid absorption more generally and has clinical implications for diseases associated with dyslipidemia.—Lobo, G. P., Hessel, S., Eichinger, A., Noy, N., Moise, A. R., Wyss, A., Palczewski, K., von Lintig, J. ISX is a retinoic acid‐sensitive gatekeeper that controls intestinal β,β‐carotene absorption and vitamin A production. FASEB J. 24, 1656–1666 (2010). www.fasebj.org

β,β-Carotene Decreases Peroxisome Proliferator Receptor γ Activity and Reduces Lipid Storage Capacity of Adipocytes in a β,β-Carotene Oxygenase 1-dependent Manner

Increasing evidence has been provided for a connection between retinoid metabolism and the activity of peroxisome proliferator receptors (Ppars) in the control of body fat reserves. Two different precursors for retinoids exist in the diet as preformed vitamin A (all-trans-retinol) and provitamin A (β,β-carotene). For retinoid production, β,β-carotene is converted to retinaldehyde by β,β-carotene monooxygenase 1 (Bcmo1). Previous analysis showed that Bcmo1 knock-out mice develop dyslipidemia and are more susceptible to diet-induced obesity. However, the role of Bcmo1 for adipocyte retinoid metabolism has yet not been well defined. Here, we showed that Bcmo1 mRNA and protein expression are induced during adipogenesis in NIH 3T3-L1 cells. In mature adipocytes, β,β-carotene but not all-trans-retinol was metabolized to retinoic acid (RA). RA decreased the expression of Pparγ and CCAAT/enhancer-binding protein α, key lipogenic transcription factors, and reduced the lipid content of mature adipocytes. This process was inhibited by the retinoic acid receptor antagonist LE450, showing that it involves canonical retinoid signaling. Accordingly, gavage of β,β-carotene but not all-trans-retinol induced retinoid signaling and decreased Pparγ expression in white adipose tissue of vitamin A-deficient mice. Our study identifies β,β-carotene as a critical physiological precursor for RA production in adipocytes and implicates provitamin A as a dietary regulator of body fat reserves.

Mammalian carotenoid-oxygenases: key players for carotenoid function and homeostasis.

Humans depend on a dietary intake of lipids to maintain optimal health. Among various classes of dietary lipids, the physiological importance of carotenoids is still controversially discussed. On one hand, it is well established that carotenoids, such as β,β-carotene, are a major source for vitamin A that plays critical roles for vision and many aspects of cell physiology. On the other hand, large clinical trials have failed to show clear health benefits of carotenoids supplementation and even suggest adverse health effects in individuals at risk of disease. In recent years, key molecular players for carotenoid metabolism have been identified, including an evolutionarily well conserved family of carotenoid-oxygenases. Studies in knockout mouse models for these enzymes revealed that carotenoid metabolism is a highly regulated process and that this regulation already takes place at the level of intestinal absorption. These studies also provided evidence that β,β-carotene conversion can influence retinoid-dependent processes in the mouse embryo and in adult tissues. Moreover, these analyses provide an explanation for adverse health effects of carotenoids by showing that a pathological accumulation of these compounds can induce oxidative stress in mitochondria and cell signaling pathways related to disease. Advancing knowledge about carotenoid metabolism will contribute to a better understanding of the biochemical and physiological roles of these important micronutrients in health and disease. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.

BCDO2 acts as a carotenoid scavenger and gatekeeper for the mitochondrial apoptotic pathway

Carotenoids and their metabolites are widespread and exert key biological functions in living organisms. In vertebrates, the carotenoid oxygenase BCMO1 converts carotenoids such as β,β-carotene to retinoids, which are required for embryonic pattern formation and cell differentiation. Vertebrate genomes encode a structurally related protein named BCDO2 but its physiological function remains undefined. Here, we show that BCDO2 is expressed as an oxidative stress-regulated protein during zebrafish development. Targeted knockdown of this mitochondrial enzyme resulted in anemia at larval stages. Marker gene analysis and staining for hemoglobin revealed that erythropoiesis was not impaired but that erythrocytes underwent apoptosis in BCDO2-deficient larvae. To define the mechanism of this defect, we have analyzed the role of BCDO2 in human cell lines. We found that carotenoids caused oxidative stress in mitochondria that eventually led to cytochrome c release, proteolytic activation of caspase 3 and PARP1, and execution of the apoptotic pathway. Moreover, BCDO2 prevented this induction of the apoptotic pathway by carotenoids. Thus, our study identifying BCDO2 as a crucial protective component against oxidative stress establishes this enzyme as mitochondrial carotenoid scavenger and a gatekeeper of the intrinsic apoptotic pathway.

Genetic ablation of the fatty acid binding protein FABP5 suppresses HER2-induced mammary tumorigenesis

The fatty acid-binding protein FABP5 shuttles ligands from the cytosol to the nuclear receptor PPARβ/δ (encoded for by Pparδ), thereby enhancing the transcriptional activity of the receptor. This FABP5/PPARδ pathway is critical for induction of proliferation of breast carcinoma cells by activated epidermal growth factor receptor (EGFR). In this study, we show that FABP5 is highly upregulated in human breast cancers and we provide genetic evidence of the pathophysiologic significance of FABP5 in mammary tumorigenesis. Ectopic expression of FABP5 was found to be oncogenic in 3T3 fibroblasts where it augmented the ability of PPARδ to enhance cell proliferation, migration, and invasion. To determine whether FABP5 is essential for EGFR-induced mammary tumor growth, we interbred FABP5-null mice with MMTV-ErbB2/HER2 oncomice, which spontaneously develop mammary tumors. FABP5 ablation relieved activation of EGFR downstream effector signals, decreased expression of PPARδ target genes that drive cell proliferation, and suppressed mammary tumor development. Our findings establish that FABP5 is critical for mammary tumor development, rationalizing the development of FABP5 inhibitors as novel anticarcinogenic drugs.

Genetics and diet regulate vitamin A production via the homeobox transcription factor ISX

Background: Dietary β-carotene is the natural precursor of vitamin A. Results: The retinoic acid-inducible homeobox transcription factor ISX controls intestinal expression of the vitamin A producing enzyme BCMO1. Conclusion: Vitamin A production is under negative feedback regulation. Significance: Large individual variability in intestinal β-carotene conversion is associated with this diet-responsive regulatory network. Low dietary intake of β-carotene is associated with chronic disease and vitamin A deficiency. β-Carotene is converted to vitamin A in the intestine by the enzyme β-carotene-15,15′-monoxygenase (BCMO1) to support vision, reproduction, immune function, and cell differentiation. Considerable variability for this key step in vitamin A metabolism, as reported in the human population, could be related to genetics and individual vitamin A status, but it is unclear how these factors influence β-carotene metabolism and vitamin A homeostasis. Here we show that the intestine-specific transcription factor ISX binds to the Bcmo1 promoter. Moreover, upon induction by the β-carotene derivative retinoic acid, this ISX binding decreased expression of a luciferase reporter gene in human colonic CaCo-2 cells indicating that ISX acts as a transcriptional repressor of BCMO1 expression. Mice deficient for this transcription factor displayed increased intestinal BCMO1 expression and produced significantly higher amounts of vitamin A from supplemental β-carotene. The ISX binding site in the human BCMO1 promoter contains a common single nucleotide polymorphism that is associated with decreased conversion rates and increased fasting blood levels of β-carotene. Thus, our study establishes ISX as a critical regulator of vitamin A production and provides a mechanistic explanation for how both genetics and diet can affect this process.

Increased adiposity in the retinol saturase-knockout mouse

The enzyme retinol saturase (RetSat) catalyzes the saturation of all‐irans‐retinol to produce (R)‐all‐trans‐13,14‐dihydroretinol. As a peroxisome pro‐liferator‐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 byRetSat control physiological processes that influence PPARγ activity and regulate lipid accumulation in mice.—Moise, A. R., Lobo, G. P., Erokwu, B., Wilson, D. L., Peck, D., Alvarez, S., Domínguez, M., Alvarez, R., Flask, C. A., de Lera, A. R., von Lintig, J., Palczewski, K. Increased adiposity in the retinol saturase‐knockout mouse. FASEB J. 24, 1261–1270 (2010). www.fasebj.org

ACTIVATION OF RETINOIC ACID RECEPTORS BY DIHYDRORETINOIDS

Vitamin A-derived metabolites act as ligands for nuclear receptors controlling the expression of a number of genes. Stereospecific saturation of the C13-C14 double bond of all-trans-retinol by the enzyme, retinol saturase (RetSat), leads to the production of (R)-all-trans-13,14-dihydroretinol. In liver and adipose tissue, expression of RetSat is controlled by peroxisome proliferator-activated receptors (PPAR) α and γ, respectively. Expression of RetSat in adipose tissue is also required for PPARγ activation and adipocyte differentiation, but the involved mechanism is poorly understood. In this study, we examined the potential of (R)-all-trans-13,14-dihydroretinol and its metabolites to control gene transcription via nuclear receptors. Using a cell-based transactivation assay to screen 25 human nuclear receptors for activation, we found that dihydroretinoids have a narrow transcriptional profile limited primarily to activation of retinoic acid receptors (RARs). Although (R)-all-trans-13,14-dihydroretinoic acid exhibited comparable potency to retinoic acid in promoting the interaction of RARs with a coactivator peptide in vitro, its potency in activating RAR-controlled genes in cell-based assays was much lower than that of retinoic acid. As an explanation for the weak RAR agonist activity of dihydroretinoids in cell-based assays, we propose that both delivery of ligand to the nucleus and RAR activation favor retinoic acid over dihydroretinoids. Discrimination between the cognate ligand, retinoic acid, and close analogs such as dihydroretinoids, occurs at multiple levels and may represent a mechanism to modulate retinoid-dependent physiological processes.

A genetic dissection of intestinal fat-soluble vitamin and carotenoid absorption

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.