Adrian Wyss

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

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

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

β-Carotene and its cleavage enzyme β-carotene-15,15`-oxygenase (CMOI) affect retinoid metabolism in developing tissues

The mammalian embryo relies on maternal circulating retinoids (vitamin A derivatives) for development. β‐Carotene is the major human dietary provitamin A. β‐Carotene‐15,15′‐oxygenase (CMOI) has been proposed as the main enzyme generating retinoid from β‐carotene in vivo. CMOI is expressed in embryonic tissues, suggesting that β‐carotene provides retinoids locally during development. We performed loss of CMOI function studies in mice lacking retinol‐binding protein (RBP), an established model of embryonic vitamin A deficiency (VAD). We show that, unexpectedly, lack of CMOI in the developing tissues further exacerbates the severity of VAD and thus the embryonic malformations of RBP−/− mice. Since β ‐carotene was not present in any of the mouse diets, we unveiled a novel action of CMOI independent from its β ‐carotene cleavage activity. We also show for the first time that CMOI exerts an additional function on retinoid metabolism by influencing retinyl ester formation via modulation of lecithin:retinol acyltransferase (LRAT) activity, at least in developing tissues. Finally, we demonstrate unequivocally that β‐carotene can serve as an alternative vitamin A source for the in situ synthesis of retinoids in developing tissues by the action of CMOI.—Kim, Y.‐K., Wassef, L., Chung, S., Jiang, H., Wyss, A., Blaner, W. S., Quadro, L. β‐Carotene and its cleavage enzyme β‐carotene‐15,15′‐oxygenase (CMOI) affect retinoid metabolism in developing tissues. FASEB J. 25, 1641–1652 (2011). www.fasebj.org

A comparison of learning and memory characteristics of young and middle-aged wild-type mice in the IntelliCage

We have tested the cognitive abilities of young (2.5 months) and middle-aged (14 months) wild-type C57Bl/6J mice in the IntelliCage, which enables automated monitoring of spontaneous and learning behaviour in a homecage-like environment. No differences were observed either in circadian activity or in performance in the novelty-induced exploration test, but middle-aged mice exhibited decreased exploratory activity overall. In the place learning test module, when mice were free to explore all corners without any negative reinforcement, young mice tended not to learn the task and performed less effectively than the middle-aged group. However, when an air-puff was administered as negative reinforcement following visits to an incorrect corner, young mice learned the task significantly better than middle-aged mice throughout the test period. Our data show that, in freely moving mice, the motivational cues for learning and retrieval of memory are age-dependent and dramatically influence learning and memory performance. Furthermore, the data reported here represent a step towards optimised cognitive test protocols when comparing young and middle-aged mice.

Vitamin E function and requirements in relation to PUFA

Vitamin E (α-tocopherol) is recognised as a key essential lipophilic antioxidant in humans protecting lipoproteins, PUFA, cellular and intra-cellular membranes from damage. The aim of this review was to evaluate the relevant published data about vitamin E requirements in relation to dietary PUFA intake. Evidence in animals and humans indicates a minimal basal requirement of 4–5 mg/d of RRR-α-tocopherol when the diet is very low in PUFA. The vitamin E requirement will increase with an increase in PUFA consumption and with the degree of unsaturation of the PUFA in the diet. The vitamin E requirement related to dietary linoleic acid, which is globally the major dietary PUFA in humans, was calculated to be 0·4–0·6 mg of RRR-α-tocopherol/g of linoleic acid. Animal studies show that for fatty acids with a higher degree of unsaturation, the vitamin E requirement increases almost linearly with the degree of unsaturation of the PUFA in the relative ratios of 0·3, 2, 3, 4, 5 and 6 for mono-, di-, tri-, tetra-, penta- and hexaenoic fatty acids, respectively. Assuming a typical intake of dietary PUFA, a vitamin E requirement ranging from 12 to 20 mg of RRR-α-tocopherol/d can be calculated. A number of guidelines recommend to increase PUFA intake as they have well-established health benefits. It will be prudent to assure an adequate vitamin E intake to match the increased PUFA intake, especially as vitamin E intake is already below recommendations in many populations worldwide.

Knockout of the Bcmo1 gene results in an inflammatory response in female lung, which is suppressed by dietary beta-carotene

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.

Monoamine reuptake inhibition and mood-enhancing potential of a specified oregano extract

A healthy, balanced diet is essential for both physical and mental well-being. Such a diet must include an adequate intake of micronutrients, essential fatty acids, amino acids and antioxidants. The monoamine neurotransmitters, serotonin, dopamine and noradrenaline, are derived from dietary amino acids and are involved in the modulation of mood, anxiety, cognition, sleep regulation and appetite. The capacity of nutritional interventions to elevate brain monoamine concentrations and, as a consequence, with the potential for mood enhancement, has not been extensively evaluated. The present study investigated an extract from oregano leaves, with a specified range of active constituents, identified via an unbiased, high-throughput screening programme. The oregano extract was demonstrated to inhibit the reuptake and degradation of the monoamine neurotransmitters in a dose-dependent manner, and microdialysis experiments in rats revealed an elevation of extracellular serotonin levels in the brain. Furthermore, following administration of oregano extract, behavioural responses were observed in mice that parallel the beneficial effects exhibited by monoamine-enhancing compounds when used in human subjects. In conclusion, these data show that an extract prepared from leaves of oregano, a major constituent of the Mediterranean diet, is brain-active, with moderate triple reuptake inhibitory activity, and exhibits positive behavioural effects in animal models. We postulate that such an extract may be effective in enhancing mental well-being in humans.

A Systematic Review of Global Alpha-Tocopherol Status as Assessed by Nutritional Intake Levels and Blood Serum Concentrations

The purpose of this study is to systematically review the published literature reporting vitamin E intake levels and serum concentrations in order to obtain a global overview of α-tocopherol status. Articles published between 2000 and 2012 were considered; 176 articles referring to 132 single studies were included. Applying an RDA (recommended daily allowance) of 15 mg/day and EAR (estimated average requirement) of 12 mg/day to all populations with a minimum age of 14 years, 82 and 61 % of mean and median data points were below the RDA and the EAR, respectively. Regarding serum concentrations, globally 13 % of the included data points were below the functional deficiency threshold concentration of 12 µmol/L, mostly for newborns and children. Several prospective observational studies suggest that a serum α-tocopherol concentration of ≥30 µmol/L has beneficial effects on human health. Of the reported study populations and subpopulations, only 21 % reached this threshold globally. This systematic review suggests that the α-tocopherol status is inadequate in a substantial part of the studied populations.