Dietary vitamin A impacts DNA methylation patterns of adipogenesis-related genes in suckling rats.

We previously showed that vitamin A supplementation in early life impacts white adipose tissue (WAT) biology. We here studied the vitamin's effects on DNA methylation of genes crucial for WAT cell development, determination and metabolism. CpG promoter methylation and mRNA expression of Pparg, Zfp423, Pcna, and Rbp4 was compared in inguinal WAT of 21-day-old rats supplemented during the suckling period with vehicle (controls) or an emulsion of vitamin A as retinyl ester (RE) or ß-carotene (BC). The methylation profile of promoters was affected by vitamin A supplementation with pronounced differences between the RE and BC groups. In the RE group, hypermethylation of the Rbp4 (at multiple CpGs) and the Pparg2 (at a specific CpG) promoters and hypomethylation of the Pcna promoter (at multiple CpGs) was observed, together with inverse changes in gene expression levels. In the BC group, hypomethylation of the Rbp4 and hypermethylation of the Pcna promoter at distinct CpGs was observed, with no effects on gene expression. In both supplemention groups, hypomethylation and increased expression was found for Zfp423. Thus, modest vitamin A supplementation in early postnatal life impacts methylation marks in developing WAT. Differential epigenetic effects of RE and BC in early life may affect adipose tissue programming activity.

Vitamin A supplementation in early life affects later response to an obesogenic diet in rats.

OBJECTIVE: To assess the influence of supplementation with a moderate dose of vitamin A in early life on adipose tissue development and the response to an obesogenic diet later in life. METHODS: During the suckling period, rat pups received a daily oral dose of retinyl palmitate corresponding to three times the vitamin A ingested daily from maternal milk. Control rats received the vehicle (olive oil). Short-term effects of treatment on gene expression and morphology of white adipose tissue (WAT) were analyzed in animals on the day after weaning (day 21). To study long-term effects, control and vitamin A-treated rats were fed, after weaning, a normal fat or a high-fat (HF) diet for 16 weeks. RESULTS: WAT of vitamin A-treated young rats (day 21) was enriched in small adipocytes with a reduced expression of adipogenic markers (peroxisome proliferator-activated receptor γ and lipoprotein lipase) and an increased cell proliferation potential as indicated by increased expression of proliferating cell nuclear antigen. Increased retinoic acid (RA)-induced transcriptional responses were present in the tissues of vitamin A-treated young rats (day 21) including WAT. Vitamin A-treated rats developed higher adiposity than control rats on a HF diet as indicated by body composition analysis and increased WAT depot mass, adipocyte diameter, WAT DNA content, leptinemia and adipose leptin gene expression. Excess adiposity gain in vitamin A-treated rats developed in the absence of changes in body weight and was attributable to excess adipocyte hyperplasia. No differences in adiposity were observed between vitamin A-treated rats and control rats on a normal fat diet. Total retinol levels in WAT of vitamin A-treated rats were elevated at weaning (day 21) and normalized by day 135 of age. CONCLUSION: Vitamin A intake in the early stages of postnatal life favors subsequent HF diet-induced adiposity gain through mechanisms that may relate to changes in adipose tissue development, likely mediated by RA.

Two common single nucleotide polymorphisms in the gene encoding beta-carotene 15,15'-monoxygenase alter beta-carotene metabolism in female volunteers.

The key enzyme responsible for beta-carotene conversion into retinal is beta-carotene 15,15'-monoxygenase (BCMO1). Since it has been reported that the conversion of beta-carotene into vitamin A is highly variable in up to 45% of healthy individuals, we hypothesized that genetic polymorphisms in the BCMO1 gene could contribute to the occurrence of the poor converter phenotype. Here we describe the screening of the total open reading frame of the BCMO1 coding region that led to the identification of two common nonsynonymous single nucleotide polymorphisms (R267S: rs12934922; A379V: rs7501331) with variant allele frequencies of 42 and 24%, respectively. In vitro biochemical characterization of the recombinant 267S + 379V double mutant revealed a reduced catalytic activity of BCMO1 by 57% (P<0.001). Assessment of the responsiveness to a pharmacological dose of beta-carotene in female volunteers confirmed that carriers of both the 379V and 267S + 379V variant alleles had a reduced ability to convert beta-carotene, as indicated through reduced retinyl palmitate:beta-carotene ratios in the triglyceride-rich lipoprotein fraction [-32% (P=0.005) and -69% (P=0.001), respectively] and increased fasting beta-carotene concentrations [+160% (P=0.025) and +240% (P=0.041), respectively]. Our data show that there is genetic variability in beta-carotene metabolism and may provide an explanation for the molecular basis of the poor converter phenotype within the population.

Molecular analysis of vitamin A formation: cloning and characterization of beta-carotene 15,15'-dioxygenases.

Beta-carotene 15,15'-dioxygenase cleaves beta-carotene into two molecules of retinal and is the key enzyme in the metabolism of carotene to vitamin A. Although the enzyme has been known for more than 40 years, all attempts to purify the protein to homogeneity or to clone its gene have failed until recently, when the successful cloning and sequencing of cDNAs encoding enzymes with beta-carotene 15,15'-dioxygenase activity from Drosophila (J. von Lintig and K. Vogt, 2000, J. Biol. Chem. 275, 11915-11920) and chicken (A. Wyss et al., 2000, Biochem. Biophys. Res. Commun. 271, 334-336) were reported. Very soon it became clear, that we have cloned two members of a new family of carotenoid cleaving enzymes. Overall homologies are very high, certain amino acid stretches almost identical. Thus, beta-carotene 15,15'-dioxygenase can be considered as evolutionarily well conserved. These findings open up wide perspectives for further analysis of this important biosynthetic pathway, concerning basic and medical research as well as biotechnological aspects related to vitamin A supply, which are discussed here.

Identification and characterization of a mammalian enzyme catalyzing the asymmetric oxidative cleavage of provitamin A.

In vertebrates, symmetric versus asymmetric cleavage of beta-carotene in the biosynthesis of vitamin A and its derivatives has been controversially discussed. Recently we have been able to identify a cDNA encoding a metazoan beta,beta-carotene-15,15'-dioxygenase from the fruit fly Drosophila melanogaster. This enzyme catalyzes the key step in vitamin A biosynthesis, symmetrically cleaving beta-carotene to give two molecules of retinal. Mutations in the corresponding gene are known to lead to a blind, vitamin A-deficient phenotype. Orthologs of this enzyme have very recently been found also in vertebrates and molecularly characterized. Here we report the identification of a cDNA from mouse encoding a second type of carotene dioxygenase catalyzing exclusively the asymmetric oxidative cleavage of beta-carotene at the 9',10' double bond of beta-carotene and resulting in the formation of beta-apo-10'-carotenal and beta-ionone, a substance known as a floral scent from roses, for example. Besides beta-carotene, lycopene is also oxidatively cleaved by the enzyme. The deduced amino acid sequence shares significant sequence identity with the beta,beta-carotene-15,15'-dioxygenases, and the two enzyme types have several conserved motifs. To establish its occurrence in different vertebrates, we then attempted and succeeded in cloning cDNAs encoding this new type of carotene dioxygenase from human and zebrafish as well. As regards their possible role, the apocarotenals formed by this enzyme may be the precursors for the biosynthesis of retinoic acid or exert unknown physiological effects. Thus, in contrast to Drosophila, in vertebrates both symmetric and asymmetric cleavage pathways exist for carotenes, revealing a greater complexity of carotene metabolism.

Filling the gap in vitamin A research. Molecular identification of an enzyme cleaving beta-carotene to retinal.

Vitamin A and its derivatives (retinoids) are essential components in vision; they contribute to pattern formation during development and exert multiple effects on cell differentiation with important clinical implications. It has been known for 50 years that the key step in the formation of vitamin A is the oxidative cleavage of beta-carotene; however, this enzymatic step has resisted molecular analysis. A novel approach enabled us to clone and identify a beta-carotene dioxygenase from Drosophila melanogaster, expressing it into the background of a beta-carotene (provitamin A)-synthesizing and -accumulating Escherichia coli strain. The carotene-cleaving enzyme, identified here for the first time on the molecular level, is the basis of the numerous branches of vitamin A action and links plant and animal carotene metabolism.

Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis.

During photomorphogenesis in higher plants, a coordinated increase occurs in the chlorophyll and carotenoid contents. The carotenoid level is under phytochrome control, as reflected by the light regulation of the mRNA level of phytoene synthase (PSY), the first enzyme in the carotenoid biosynthetic pathway. We investigated PSY protein levels, enzymatic activity and topological localization during photomorphogenesis. The results revealed that PSY protein levels and enzymatic activity increase during de-etiolation and that the enzyme is localized at thylakoid membranes in mature chloroplasts. However, under certain light conditions (e.g., far-red light) the increases in PSY mRNA and protein levels are not accompanied by an increase in enzymatic activity. Under those conditions, PSY is localized in the prolamellar body fraction in a mostly enzymatically inactive form. Subsequent illumination of dark-grown and/or in far-red light grown seedlings with white light causes the decay of these structures and a topological relocalization of PSY to developing thylakolds which results in its enzymatic activation. This light-dependent mechanism of enzymatic activation of PSY in carotenoid biosynthesis shares common features with the regulation of the NADPH:protochlorophyllide oxidoreductase, the first light-regulated enzyme in chlorophyll biosynthesis. The mechanism of regulation described here may contribute to ensuring a spatially and temporally coordinated increase in both carotenoid and chlorophyll contents.

Light-dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and is mediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings.

In chloroplasts, carotenoids are essential pigments involved in photosynthesis. During-photomorphogenesis, a coordinated increase in the amounts of chlorophylls and carotenoids, in conjugation with other components, leads to the formation of a functional photosynthetic apparatus. To investigate the regulation of carotenoid biosynthesis during this process at the molecular level, GGPS, PSY and PDS cDNAs have been cloned from white mustard (Sinapis alba L). GGPS encodes a key enzyme in plastid isoprenoid metabolism, while the products of PSY and PDS catalyse the subsequent steps in carotenoid biosynthesis. Due to the low mRNA levels of the genes involved, the use of a RT-PCR protocol was necessary to measure gene expression during photomorphogenesis. With light, there is an up-regulation of PSY expression, the first gene within the carotenoid biosynthetic pathway, while PDS and GGPS expression levels remain constant. Treatment with different light qualities reveals a phytochrome-mediated regulation of PSY expression in developing white mustard seedlings. To obtain more detailed information on the light-regulation, Arabidopsis thaliana wild-type and phytochrome mutants were utilized. Continuous far-red and red light both increase the expression of PSY in wild-type seedlings, demonstrating that both light-labile and light-stable phytochromes are involved in PSY regulation. The response to far-red light is completely abolished in the phyA mutant, showing that PHYA mediates the increase in PSY transcript levels under these light conditions. In the phyB mutant, the red light response is normal, indicating that PSY expression is not controlled by PHYB but by other light-stable phytochromes. Measurement of chlorophylls and carotenoids under the same light regimes shows that the up-regulation of PSY expression does not necessarily result in an increase of the carotenoid content. Only those light conditions which allow chlorophyll biosynthesis lead to a significant increase of the carotenoid content. Therefore, it is proposed that up-regulation of PSY mRNA levels leads to an increased capacity for the formation of carotenoids. However, this only takes place under light conditions leading to protochlorophyllide photoconversion.

Chloroplast import of four carotenoid biosynthetic enzymes in vitro reveals differential fates prior to membrane binding and oligomeric assembly.

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-molecular-mass 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 high-molecular-mass complexes of imported carotenogenic enzymes. Finally, it was inferred that a membrane-bound regulatory factor plays a decisive role in membrane-binding.

Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis.

Rice (Oryza sativa L.), the major food staple for more than two billion people, contains neither beta-carotene (provitamin A) nor C40 carotenoid precursors thereof in its endosperm. To improve the nutritional value of rice, genetic engineering was chosen as a means to introduce the ability to make beta-carotene into rice endosperm tissue. Investigation of the biochemical properties of immature rice endosperm using [14C]-labelled substrates revealed the presence of geranyl geranyl diphosphate, the C20 general isoprenoid precursor necessary for C40 carotenoid biosynthesis. Phytoene synthase, which condenses two molecules of geranyl geranyl diphosphate, is the first of four specific enzymes necessary for beta-carotene biosynthesis in plants. Therefore, the Japonica rice model variety Taipei 309 was transformed by microprojectile bombardment with a cDNA coding for phytoene synthase from daffodil (Narcissus pseudonarcissus) under the control of either a constitutive or an endosperm-specific promoter. In transgenic rice plants, the daffodil enzyme is active, as measured by the in vivo accumulation of phytoene in rice endosperm. Thus, it is demonstrated for the first time that it is in principle possible to engineer a critical step in provitamin A biosynthesis in a non-photosynthetic, carotenoid-lacking plant tissue. These results have important implications for long-term prospects of overcoming worldwide vitamin A deficiency.

Phytoene synthase from Narcissus pseudonarcissus: functional expression, galactolipid requirement, topological distribution in chromoplasts and induction during flowering.

A cDNA coding for the carotenoid biosynthetic enzyme phytoene synthase was cloned from a Narcissus pseudonarcissus flower cDNA library, and the corresponding protein was overexpressed in insect cells using the baculovirus lipofection system. The full-length overexpressed enzyme exhibited very reduced catalytic activity compared with an overexpressed N-truncated form, with its transit sequence removed by site-directed mutagenesis. The shortened form readily bound quantitatively to lipid bilayers. Although it was active with liposomes prepared from plastid lipids, with phospholipid liposomes it was not, even though association took place. In this latter case, free galactose was capable of substituting for galactolipids, resulting in enzymatic activity. It is concluded that galactolipids are involved in catalytic activity, but do not serve as a membrane anchor. Antibodies raised against the recombinant enzyme made it possible to distinguish between a membrane-bound and a soluble, protein-complexed inactive form of phytoene synthase, present in the chromoplast stroma. These findings and data on phytoene synthase mRNA and protein expression presented here are discussed in terms of a possible regulatory role in color formation during chromoplast (flower) development.

A novel, soluble form of phytoene desaturase from Narcissus pseudonarcissus chromoplasts is Hsp70-complexed and competent for flavinylation, membrane association and enzymatic activation.

A cDNA coding for the carotenoid biosynthetic enzyme phytoene desaturase from Narcissus pseudonarcissus was cloned and the corresponding protein expressed in insect cells using the baculovirus system. Polyclonal antibodies raised against the recombinant protein allowed the detection of soluble and tightly membrane-bound populations of phytoene desaturase in the chromoplasts isolated from petals. The soluble form is enzymatically inactive and a constituent of a larger Hsp 70-containing protein complex in the stroma, whereas the membrane-bound form is functional. In vitro, the soluble form is able to associate on to/into protein-free liposomal membranes made from chromoplast lipids, thereby gaining activity by binding added flavine adenine dinucleotide (FAD). Once bound to membranes, activated phytoene desaturase works independently of any added FAD, employing membrane-bound electron acceptors. FAD, however, exerts no positive effect on the membrane-association process. Its role is confined to enzymatic activation. Although carotenoid accumulation is strongly induced during flower development, only very low concentrations of phytoene desaturase transcripts are detectable, while the corresponding protein accumulates in low, but measurable amounts, appearing in soluble and membrane-bound states. Post-transcriptional mechanisms contribute significantly to carotenoid accumulation, as do factors determining the enzymatic activity of phytoene desaturase, for example by influencing the redox-state of membrane-bound electron acceptors.

Ti plasmid-encoded octopine and nopaline catabolism in Agrobacterium: specificities of the LysR-type regulators OccR and NocR, and protein-induced DNA bending.

The occ and noc regions in octopine and nopaline Ti plasmids, respectively, are responsible for the catabolism of octopine and nopaline in Agrobacterium. The functions are activated in the presence of the opines by OccR and NocR, two related regulatory proteins, and the promoters contain common sequence motifs. We have investigated heterologous interactions between the regulators and the promoters. Previous experiments using all possible heterologous combinations of opines, regulators, and promoters in vivo had demonstrated that only the combination of nopaline, NocR, and the occ promoter led to limited promoter activation. We now show that OccR and NocR bind to the heterologous promoters in vitro and in vivo. The weak or non-existent promoter activation actually observed could be explained by the assumption that OccR and NocR use different activation mechanisms; we investigated protein-induced DNA bending because of reports that the two regulators differ in this respect. Analysis with a bending vector showed that both OccR and NocR induced a DNA bend that is relaxed in the presence of the respective opine. The data suggest that subtle differences in regulator/promoter interactions are responsible for the inactivity of the heterologous combinations. Investigations with a chimeric NocR/OccR protein indicated that it induced a DNA bend in both promoters. No opine-induced relaxation was detectable with the hybrid, and the inducible promoter was not activated. These findings suggest that bend relaxation may be an integral part of promoter activation.

Opine-regulated promoters and LysR-type regulators in the nopaline (noc) and octopine (occ) catabolic regions of Ti plasmids of Agrobacterium tumefaciens.

Essential steps in the uptake and catabolism of the plant tumor metabolites nopaline and octopine in Agrobacterium spp. are performed by proteins encoded in the nopaline catabolic (noc) and octopine catabolic (occ) regions of Ti plasmids. We investigated the opine activation of the genes by using (i) promoter studies of Agrobacterium spp. and (ii) analysis of the promoter interaction with the regulatory proteins NocR (noc) and OccR (occ). The noc region contained two nopaline-induced promoters (Pi1[noc] and Pi2[noc]) and one autogenously regulated promoter (Pr [control of NocR expression]). Pi2 and Pr overlapped and were divergently oriented (Pi2 [noc]). DNA binding studies and DNase I footprints indicated that NocR bound specifically to single binding sites in Pi1[noc] and Pi2/Pr[noc] and that Pi2 and Pr were regulated from the same binding site. The binding was independent of the inducer nopaline, and nopaline caused small changes in the footprint. The promoters in the noc and occ regions shared sequence motif and contained the sequence T-N11-A, which is characteristic for LysR-type-regulated promoters. The occ region contained one octopine-induced and one autogenously regulated promoter (Pi/Pr[occ]) in the same arrangement as Pi2/Pr[noc] in the noc region. Promoter deletions indicated that sequences flanking the OccR binding site determined the extent of induction, although they did not bind OccR. The promoter bound OccR in the absence and presence of octopine. The opine caused a change in the mobility of the DNA-protein complex with the complete promoter. The resected fragments did not reveal this opine-induced shift, and it was also not detectable with the DNA-NocR complexes with the two promoters of the noc region.

Opine transport genes in the octopine (occ) and nopaline (noc) catabolic regions in Ti plasmids of Agrobacterium tumefaciens.

The occ and noc regions of octopine and nopaline Ti plasmids in Agrobacterium tumefaciens are responsible for the catabolic utilization of octopine and nopaline, respectively. Opine-inducible promoters, genes for regulatory proteins and for catabolic enzymes, had been identified in previous work. However, both regions contained additional DNA stretches which were under the control of opine-inducible promoters, but the functions were unknown. We investigated these stretches by DNA sequence and functional analyses. The sequences showed that both of the catabolic regions contain a set of four genes which are transcribed in the same direction. The occ and noc region genes are related, but the arrangement of the genes is different. The deduced polypeptides are related to those of binding protein-dependent transport systems of basic amino acids in other bacteria. The comparison suggested that three of the polypeptides are located in the membrane and that one is a periplasmic protein. We constructed cassettes which contained either the putative transport genes only or the complete occ or noc region; all constructs, however, included the elements necessary for opine-induced expression of the genes (the regulatory gene and the inducible promoters). Uptake studies with 3H-labelled octopine showed that the putative transport genes in the occ region code for octopine uptake proteins. The corresponding studies with 3H-labelled nopaline and the noc region cassettes indicated that the uptake of nopaline requires the putative transport genes and additional functions from the left part of the noc region.

Positive regulators of opine-inducible promoters in the nopaline and octopine catabolism regions of Ti plasmids.

The noc and occ regions of nopaline and octopine Ti plasmids in Agrobacterium tumefaciens contain genes for the catabolism of nopaline and octopine, respectively. We investigated the transcriptional organization and regulation of both regions. The noc region of pTiC58 contains two nopaline-inducible promoters, and one octopine-inducible promoter was identified in the occ region of pTiAch5. All three promoters are positively regulated in trans by constitutively expressed genes localized at the right end of the regions. The DNA sequence analysis of these parts revealed genes coding for related proteins (35.6% identity). The two polypeptides share significant similarity with a family of other positive gene regulators, and both contain a protein motif ("LysR" signature) that is characteristic for the DNA binding domain in these polypeptides. These proteins are the only Ti plasmid functions necessary for the activation of the opine-induced promoters. We propose the names nocR and occR for the regulator genes in the noc and in the occ regions.