Jaume Amengual

CMO1 Deficiency Abolishes Vitamin A Production from β-Carotene and Alters Lipid Metabolism in Mice

Carotenoids are currently investigated regarding their potential to lower the risk of chronic disease and to combat vitamin A deficiency in humans. These plant-derived compounds must be cleaved and metabolically converted by intrinsic carotenoid oxygenases to support the panoply of vitamin A-dependent physiological processes. Two different carotenoid-cleaving enzymes were identified in mammals, the classical carotenoid-15,15′-oxygenase (CMO1) and a putative carotenoid-9′,10′-oxygenase (CMO2). To analyze the role of CMO1 in mammalian physiology, here we disrupted the corresponding gene by targeted homologous recombination in mice. On a diet providing β-carotene as major vitamin A precursor, vitamin A levels fell dramatically in several tissues examined. Instead, this mouse mutant accumulated the provitamin in large quantities (e.g. as seen by an orange coloring of adipose tissues). Besides impairments in β-carotene metabolism, CMO1 deficiency more generally interfered with lipid homeostasis. Even on a vitamin A-sufficient chow, CMO1-/- mice developed a fatty liver and displayed altered serum lipid levels with elevated serum unesterified fatty acids. Additionally, this mouse mutant was more susceptible to high fat diet-induced impairments in fatty acid metabolism. Quantitative reverse transcription-PCR analysis revealed that the expression of peroxisome proliferator-activated receptor γ-regulated marker genes related to adipogenesis was elevated in visceral adipose tissues. Thus, our study identifies CMO1 as the key enzyme for vitamin A production and provides evidence for a role of carotenoids as more general regulators of lipid metabolism.

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

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.

Beta-Carotene Reduces Body Adiposity of Mice via BCMO1

Evidence from cell culture studies indicates that β-carotene-(BC)-derived apocarotenoid signaling molecules can modulate the activities of nuclear receptors that regulate many aspects of adipocyte physiology. Two BC metabolizing enzymes, the BC-15,15′-oxygenase (Bcmo1) and the BC-9′,10′-oxygenase (Bcdo2) are expressed in adipocytes. Bcmo1 catalyzes the conversion of BC into retinaldehyde and Bcdo2 into β-10′-apocarotenal and β-ionone. Here we analyzed the impact of BC on body adiposity of mice. To genetically dissect the roles of Bcmo1 and Bcdo2 in this process, we used wild-type and Bcmo1 -/- mice for this study. In wild-type mice, BC was converted into retinoids. In contrast, Bcmo1-/- mice showed increased expression of Bcdo2 in adipocytes and β-10′-apocarotenol accumulated as the major BC derivative. In wild-type mice, BC significantly reduced body adiposity (by 28%), leptinemia and adipocyte size. Genome wide microarray analysis of inguinal white adipose tissue revealed a generalized decrease of mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) target genes. Consistently, the expression of this key transcription factor for lipogenesis was significantly reduced both on the mRNA and protein levels. Despite β-10′-apocarotenoid production, this effect of BC was absent in Bcmo1-/- mice, demonstrating that it was dependent on the Bcmo1-mediated production of retinoids. Our study evidences an important role of BC for the control of body adiposity in mice and identifies Bcmo1 as critical molecular player for the regulation of PPARγ activity in adipocytes

Two Carotenoid Oxygenases Contribute to Mammalian Provitamin A Metabolism

Background: Mammalian genomes encode two carotenoid oxygenases, but their contributions to vitamin A homeostasis remain undefined. Results: Mammals employ symmetric and eccentric cleaving carotenoid oxygenases to convert different provitamin A carotenoids to vitamin A. Conclusion: Both carotenoid oxygenases contribute to vitamin A production. Significance: Carotenoids are the major source for vitamin A in the human diet. 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.

Lecithin:retinol acyltransferase is critical for cellular uptake of vitamin A from serum retinol-binding protein.

Background: Cellular uptake of retinol bound to its serum binding protein depends on a cell surface receptor. Results: Functional coupling of this receptor with lecithin:retinol acyltransferase is required for the regulated cellular uptake of retinol. Conclusion: The lecithin: retinol acyltransferase is critical for retinol uptake and homeostasis. Significance: Blood retinol homeostasis is associated with blinding retinopathies and diabetes. Vitamin A (all-trans-retinol) must be adequately distributed within the mammalian body to produce visual chromophore in the eyes and all-trans-retinoic acid in other tissues. Vitamin A is transported in the blood bound to retinol-binding protein (holo-RBP), and its target cells express an RBP receptor encoded by the Stra6 (stimulated by retinoic acid 6) gene. Here we show in mice that cellular uptake of vitamin A from holo-RBP depends on functional coupling of STRA6 with intracellular lecithin:retinol acyltransferase (LRAT). Thus, vitamin A uptake from recombinant holo-RBP exhibited by wild type mice was impaired in Lrat−/− mice. We further provide evidence that vitamin A uptake is regulated by all-trans-retinoic acid in non-ocular tissues of mice. When in excess, vitamin A was rapidly taken up and converted to its inert ester form in peripheral tissues, such as lung, whereas in vitamin A deficiency, ocular retinoid uptake was favored. Finally, we show that the drug fenretinide, used clinically to presumably lower blood RBP levels and thus decrease circulating retinol, targets the functional coupling of STRA6 and LRAT to increase cellular vitamin A uptake in peripheral tissues. These studies provide mechanistic insights into how vitamin A is distributed to peripheral tissues in a regulated manner and identify LRAT as a critical component of this process.

Retinoic Acid Treatment Enhances Lipid Oxidation and Inhibits Lipid Biosynthesis Capacities in the Liver of Mice

Vitamin A, mainly as retinoic acid (RA), is known to affect the development and function of adipose tissues. Treatment with RA reduces body weight and adiposity independent of changes in food intake in mice. Lipid metabolism in liver can have a major impact on whole body adiposity. The aim of this work was to investigate the effects of an in vivo treatment with RA on hepatic lipid metabolism in mice. Adult, standard diet-fed mice were treated with different doses of all-trans RA or vehicle (subcutaneous injection) for 4 days before sacrifice. Food intake and body weight changes during treatment were determined, as well as adiposity, liver composition, levels of circulating metabolites and lipoproteins and expression levels of key mRNA species in liver following sacrifice. RA treatment resulted in reduced body weight and adiposity, as expected. In the liver, RA treatment triggered an increase in the mRNA expression levels of peroxisome proliferator-activated receptor alpha, retinoid X receptor alpha, uncoupling protein 2, liver-type carnitine palmitoyltransferase 1, and carnitine/acylcarnitine carrier, and a reduction in the mRNA expression levels of sterol regulatory element binding protein 1c and fatty acid synthase. Consistent with the changes in gene expression, hepatic triacylglycerol content and circulating VLDL fraction were reduced and levels of circulating ketone bodies increased after RA treatment. These results point to a capacity of active vitamin A forms to shift liver lipid metabolism in vivo towards increased catabolism and reduced lipogenesis. These effects might contribute to the reduction of adiposity brought about by RA treatment.

Retinoic Acid Treatment Increases Lipid Oxidation Capacity in Skeletal Muscle of Mice

Objective: All‐trans retinoic acid (ATRA), a carboxylic form of vitamin A, favors in mice a mobilization of body fat reserves that correlates with an increment of oxidative and thermogenic capacity in adipose tissues. The objective of this study has been to investigate the effect of ATRA treatment on skeletal muscle capacity for fatty‐acid catabolism.

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.