BBA - Molecular Basis of Disease (v.1740, #2)

Carotenoids: risk or benefit for health by Aldona Dembinska-Kiec (93-94).
Keywords: Carotenoid; Cell proliferation; Differentiation;

Challenges to understanding and measuring carotenoid bioavailability by Richard Martin Faulks; Susan Southon (95-100).
Carotenoids are an excellent example of where poor understanding of food structure, complexity of behaviour during digestion, and inter-individual differences in response, lead to misinterpretation of study results. Four challenges associated with understanding and measuring carotenoid bioavailability are discussed: release of carotenoids from food structure and processing into an absorbable form (bioaccessibility), passage of carotenoids from gut lumen into the body (absorption), interpreting plasma response and inter-individual variation.Bioaccessibility of carotenoids is governed by characteristics of the food matrix, which affect the efficiency of physical, enzymic and chemical digestion. Carotenoids used as colorants are likely to be better absorbed because of the form in which they are dispersed in food. Extent of absorption of carotenoid supplements will depend on the proximity of dosing to the consumption of a fat-containing meal. Release of carotenoids from food plants occurs only when the plant cell is fractured and this occurs only during food preparation, processing and/or mastication, not during digestion. Following release from the food matrix, the major limiting factor is solubility of carotenoids in digesta.Absorption studies are best carried out by measuring chylomicron carotenoid excursion, with modelling of chylomicron turnover rate. In this way, inter-individual differences in lipoprotein metabolism can, in part, be taken into account before formulating conclusions on the rate and extent of absorption.
Keywords: Carotenoid; Bioavailability; Digestion; Absorption;

Bioactivity and protective effects of natural carotenoids by Wilhelm Stahl; Helmut Sies (101-107).
Carotenoids comprise a class of natural fat-soluble pigments which are found in numerous fruits and vegetables. The consumption of a diet rich in carotenoids has been epidemiologically correlated with a lower risk for several diseases. The antioxidant activity of carotenoids and biochemical properties influencing signaling pathways have been discussed as basic mechanisms of prevention. Conflicting data from intervention studies with β-carotene to prevent cancers and cardiovascular disorders have challenged the concept. However, there is convincing evidence that carotenoids are important components of the antioxidant network. Photooxidative damage is suggested to be involved in the pathobiochemistry of several diseases affecting the skin and the eye, and carotenoids may protect light-exposed tissues. Lutein and zeaxanthin are the predominant carotenoids of the retina and are considered to act as photoprotectants preventing retinal degeneration. The unique distribution, localization and high levels of both carotenoids within the macula lutea as well as their physicochemical properties make them suitable candidates for photoprotection. β-Carotene is used as an oral sun protectant for the prevention of sunburn and has been shown to be effective either alone or in combination with other carotenoids or antioxidant vitamins. Protective effects are also achieved with a diet rich in lycopene.
Keywords: Carotenoid; Prevention; Disease; Signaling; Antioxidant;

Carotenoids as modulators of lipid membrane physical properties by Wiesław I. Gruszecki; Kazimierz Strzałka (108-115).
Carotenoids are a group of pigments present both in the plant and animal kingdoms, which play several important physiological functions. The protection against active oxygen species, realised via the quenching of excited states of photosensitising molecules, quenching of singlet oxygen and scavenging of free radicals, is one of the main biological functions of carotenoids. Several recent research indicate that the protection of biomembranes against oxidative damage can be also realised via the modification of the physical properties of the lipid phase of the membranes. This work presents an overview of research on an effect of carotenoids on the structural and dynamic properties of lipid membranes carried out with the application of different techniques such as Electron Paramagnetic Resonance, Nuclear Magnetic Resonance, Differential Scanning Calorimetry, X-ray diffractometry, monomolecular layer technique and other techniques. It appears that, in most cases, polar carotenoids span lipid bilayer and have their polar groups anchored in the opposite polar zones of the membrane. Owing to the van der Waals interactions of rigid rod-like molecules of carotenoid and acyl chains of lipids, pigment molecules rigidify the fluid phase of the membranes and limit oxygen penetration to the hydrophobic membrane core susceptible to oxidative degradation.
Keywords: Carotenoid; Membrane;

There is growing evidence that high levels of the macular xanthophyll carotenoids lutein and zeaxanthin may be protective against visual loss due to age-related macular degeneration, but the actual mechanisms of their protective effects are still poorly understood. We have recently purified, identified and characterized a pi isoform of glutathione S-transferase (GSTP1) as a zeaxanthin-binding protein in the macula of the human eye which specifically and saturably binds to the two forms of zeaxanthin endogenously found in the foveal region. In this report, we studied the synergistic antioxidant role of zeaxanthin and GSTP1 in egg yolk phosphatidylcholine (EYPC) liposomes using hydrophilic 2,2′-azobis(2-methyl-propionamidine) dihydrochloride (AAPH) and lipophilic 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN) as lipid peroxyl radical generators. The two zeaxanthin diastereomers displayed synergistic antioxidant effects against both azo lipid peroxyl radical generators when bound to GSTP1. In the presence of GSTP1, nondietary (3R,3′S-meso)-zeaxanthin was observed to be a better antioxidant than dietary (3R,3′R)-zeaxanthin. This effect was found to be independent of the presence of glutathione. Carotenoid degradation profiles indicated that the zeaxanthin diastereomers in association with GSTP1 were more resistant to degradation which may account for the synergistic antioxidant effects.
Keywords: Zeaxanthin; Binding protein; Antioxidant; Lipid; Carotenoid;

Towards a better understanding of carotenoid metabolism in animals by Johannes von Lintig; Susanne Hessel; Andrea Isken; Cornelia Kiefer; Johanna M. Lampert; Olaf Voolstra; Klaus Vogt (122-131).
Vitamin A 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. All naturally occurring vitamin A derives by enzymatic oxidative cleavage from carotenoids with provitamin A activity. To become biologically active, these plant-derived compounds must first be absorbed, then delivered to the site of action in the body, and metabolically converted to the real vitamin. Recently, molecular players of this pathway were identified by the analysis of blind Drosophila mutants. Similar genome sequences were found in vertebrates. Subsequently, these homologous genes were cloned and their gene products were functionally characterized. This review will summarize the advanced state of knowledge about the vitamin A biosynthetic pathway and will discuss biochemical, physiological, developmental and medical aspects of carotenoids and their numerous derivatives.
Keywords: Vitamin A; Carotenoid metabolism in animal; bco; bco2; rpe65; Retinoic acid signaling;

Beta-carotene uptake and metabolism in human lung bronchial epithelial cultured cells depending on delivery vehicle by Ana M. Rodríguez; Sebastián Sastre; Joan Ribot; Andreu Palou (132-138).
Beta-carotene (BC) could have a protective or pro-carcinogenic role in lung cancer, and cell culture systems are important to evaluate it. Nevertheless, the delivery of the hydrophobic BC to cells is difficult. Different vehicles have been used such as liposomes, tetrahydrofuran and serum lipoproteins, but presenting different problems. Water dispersible beadlets containing BC are a good choice and can produce the greatest BC uptake when compared to the above vehicles, but other beadlet components could alter the results. Dimethylsulfoxide (DMSO) could be a good alternative since it has low toxicity and it enhances the penetration of substances across biologic membranes. We aimed to characterize an appropriate model for delivering all-trans-BC to lung cells in culture and knowing its metabolism. All-trans-BC 5 μM was administered to BEAS-2B cells in beadlets or DMSO, and medium and cell samples were taken at different times. The levels of BC and its main isomers and metabolites were determined by HPLC. All-trans-BC reached the same levels in the medium (about 3.5 μM) either when supplied in beadlets or in DMSO, and, with beadlets, 13-cis-BC was also detected. However the amount of all-trans-BC taken up by the cells was the triple when delivered by DMSO. With both vehicles, intracellular all-trans-BC levels reached its maximum after 24 h of treatment, remaining equal after 72 h. The 9-cis and 13-cis isomers of BC, and oxidized metabolites, were also detected in the cells although in smaller proportion than all-trans-BC, especially with DMSO. An LDH assay did not suggest toxicity of beadlets, DMSO or BC itself. In conclusion, DMSO seems the most appropriate vehicle for delivering BC to lung cells in vitro, and we present a model that allows studying the effects of BC and its metabolism in the lung human BEAS-2B cell line.
Keywords: Beta-carotene; Lung cancer; BEAS-2B; Dimethylsulfoxide; Beadlet;

Beta-carotene and the application of transcriptomics in risk–benefit evaluation of natural dietary components by Jaap Keijer; Annelies Bunschoten; Andreu Palou; Nicole L.W. Franssen-van Hal (139-146).
Beta-carotene is a natural food component that is present in fruits and vegetables and is also used as a food colorant and a supplement. Beta-carotene is an anti-oxidant and a source of vitamin A. It is endowed with health beneficial properties, but a number of studies showed that with high intakes it may increase the risk for lung cancer in at risk individuals (heavy smokers, asbestos workers and alcohol users). To establish the window of benefit, it is necessary to identify early markers of effect and to obtain insight in the mechanism of action of beta-carotene, in the absence and presence of environmental risk factors. Genomics technologies are well suited to dissect the mechanisms of action and identify the markers of effect. Human cell lines can be used to analyse the effects of beta-carotene, but exposure studies with beta-carotene show that cell lines display a widely variant behaviour, which hampers translation to the in vivo situation in humans. Alternatively, animal studies can be used. Especially the ferret seems to be a good model, but little sequence information of this species is available. However, heterologous hybridization on human cDNA seems possible and provides and a new tool for molecular analysis of health effects of beta-carotene.
Keywords: Beta-carotene; Microarray; Ferret; Cell lines; Lung; Colon;

Carotenoids are highly bioactive dietary compounds that have the potential to have significant effects on human health. It is becoming increasingly clear that the various biological effects that carotenoids exert could be driven via a number of different mechanisms. These include direct pro- and antioxidant effects, redox sensitive cell signalling, vitamin A signalling pathways and other as yet unidentified mechanisms. This article provides an overview of the known effects of carotenoids and discusses the use of model systems and functional genomic approaches further to elucidate their modes of action.
Keywords: Carotenoid; β-Carotene; Vitamin A; Gene expression; Functional genomics; Model system;

Gene expression profiling identifies retinoids as potent inducers of macrophage lipid efflux by Thomas Langmann; Gerhard Liebisch; Christoph Moehle; Rainer Schifferer; Rania Dayoub; Susanne Heiduczek; Margot Grandl; Ashraf Dada; Gerd Schmitz (155-161).
Vitamin A and its naturally occurring derivatives 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (ATRA) exert a variety of biological effects including immunomodulation, growth, differentiation, and apoptosis of normal and neoblastic cells. In order to directly study the effects of these retinoids on macrophage gene expression and lipid metabolism, primary human monocytes and in vitro differentiated macrophages were stimulated with β-carotene, 9-cis RA, and ATRA and global gene expression profiles were analyzed by Affymetrix DNA-microarrays and differentially regulated genes were verified by quantitative TaqMan RT-PCR. Among others, we have identified a strong up-regulation of a cluster of genes involved in cholesterol metabolism including apolipoproteins (apoC-I, apoC-II, apoC-IV, apoE), the scavenger receptor CD36, steroid-27-hydroxylase (CYP27A1), liver X receptor α (LXRα), and ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1). Since the CYP27A1 gene displayed the strongest up-regulation on the mRNA level, we cloned various deletion constructs of the promoter region and analyzed the response to retinoids in macrophages. Thereby, a novel retinoic acid-responsive element could be located within 191 bp of the proximal CYP27A1 promoter. To further assess the functional consequences of retinoid receptor action, we carried out phospholipid and cholesterol efflux assays. We observed a strong induction of apoA-I-dependent lipid efflux in stimulated macrophages, implicating an important role for retinoids in cellular functions of macrophages.
Keywords: Retinoid; Macrophage; Lipid efflux; CYP27A1; ABC transporter;

β-carotene is the major carotenoid occurring in the human diet and in the human organism. Besides its function as pro-vitamin A, β-carotene has been shown to be an activator of the human pregnan X receptor (PXR). PXR is mainly expressed in the liver/intestine and an inducer of enzymes involved in phase I, II and III metabolism. This review is focused on the evaluation of physiological and nutritional relevance of β-carotene as an inducer of phase I enzymes in the human organism via PXR-mediated mechanisms. Beneficial and detrimental effects of β-carotene on xenobiotica metabolism and metabolism of various other derivatives will be discussed.
Keywords: β-carotene; Carotenoid; Retinoid; Retinoic; Vitamin A; PXR; CYP3A;

Virtually all human tumors are deficient in gap junctional communication (GJC) and the restoration of GJC by forced expression of connexins reduces indices of neoplasia. The expression of connexin 43 (Cx43) is upregulated by cancer-preventive retinoids and carotenoids which correlates with the suppression of carcinogen-induced transformation in 10T1/2 cells. However, the molecular mechanism for upregulated expression is poorly understood. The retinoic acid receptor antagonist, Ro 41-5253, suppressed retinoid-induced Cx43 protein expression in 10T1/2 cells and the induction of a Cx43 luciferase reporter construct in F9 cells, but did not suppress protein expression or reporter activity induced by the non-pro-vitamin A carotenoid astaxanthin. In contrast, Cx43 induction by astaxanthin, but not by a RAR-specific retinoid, was inhibited by GW9662, a PPAR-γ antagonist. Neither compound required protein synthesis for the induction of Cx43 mRNA, nor was the 5.0 h half-life of Cx43 mRNA altered, indicating direct transcriptional activation. The responsive region was found within −158 bp and +209 bp of the transcription start site. Site directed mutagenesis of a GC-box in this region increased basal levels of transcription and loss of retinoid responsiveness. Simultaneous treatment with a retinoid and β-carotene or astaxanthin resulted in supra-additive Cx43 expression, again indicating separate mechanisms of gene regulation.

β-Carotene interaction with NNK in the AJ-mouse model: Effects on cell proliferation, tumor formation and retinoic acid responsive genes by Regina Goralczyk; Karin Wertz; Barbara Lenz; Georges Riss; Petra Buchwald Hunziker; Brad Geatrix; Claude-P. Aebischer; Heinrich Bachmann (179-188).
We studied the influence of β-carotene on the tobacco smoke carcinogen 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumor development in the A/J-mouse model. The normally low β-carotene absorption was facilitated with a diet enriched in fat and bile salt, resulting in plasma and lung tissue levels similar to humans.β-Carotene enhanced NNK-induced early bronchial cell proliferation, however, this effect was not predictive for later tumor development. Tumor multiplicity was not significantly affected by β-carotene, neither in carcinogen-initiated nor in uninitiated mice, and regardless of dose and time point of supplementation during tumor development. RARβ isoform and CYP26 gene expression levels analyzed by quantitative RT-PCR were weakly, but significantly, inversely correlated and showed evidence for altered retinoid signaling and catabolism in the lungs of NNK-initiated, β-carotene supplemented mice. However, this interaction did not translate into enhanced tumor multiplicity. These results indicate that impaired retinoid signaling is not likely a key factor in lung tumorigenesis in this mouse model.
Keywords: β-Carotene; Lung cancer; A/J-mouse; NNK; RARβ; CYP26;

Different effect of beta-carotene on proliferation of prostate cancer cells by Joanna Dulińska; Dorota Gil; Jacek Zagajewski; Jadwiga Hartwich; Marek Bodzioch; Aldona Dembińska-Kieć; Thomas Langmann; Gerd Schmitz; Piotr Laidler (189-201).
It was shown that high doses of beta-carotene (>30 μM) decrease proliferation of prostate cancer cells in vitro. However, it is rather doubtful whether such concentration of beta-carotene is really accessible at cellular level. We studied the effect of 3 and 10 μM beta-carotene on proliferation and gene expression in LNCaP and PC-3 prostate cancer cell lines. Beta - carotene–more efficiently absorbed from medium by androgen-sensitive LNCaP cells – increased proliferation of LNCaP cells whereas it had weaker effect on PC-3 cells. Initial global analysis of expression of genes in both cell lines treated with 10 μM beta-carotene (Affymetrix HG-U133A) showed remarkable differences in number of responsive genes. Their recognition allows for conclusion that differences between prostate cancer cell lines in response to beta-carotene treatment are due to various androgen sensitivities of LNCaP and PC-3 cells. Detailed analysis of expression of selected genes in beta-carotene treated LNCaP cells at the level of mRNA and protein indicated that the observed increase of proliferation could have been the result of slight induction of a few genes affecting proliferation (c-myc, c-jun) and apoptosis (bcl-2) with no significant effect on major cell cycle control genes (cdk2, RB, E2F-1).
Keywords: Beta-carotene; Prostate cancer; Proliferation; Gene expression; Cell cycle;

Role of lycopene and tomato products in prostate health by Maria Stacewicz-Sapuntzakis; Phyllis E. Bowen (202-205).
Epidemiological evidence associating the decreased risk of prostate cancer with frequent consumption of tomato products inspired us to conduct a small intervention trial among patients diagnosed with prostate adenocarcinoma. Tomato sauce pasta was consumed daily for 3 weeks before their scheduled prostatectomy, and biomarkers of tomato intake, prostate cancer progression and oxidative DNA damage were followed in blood and the available prostate tissue. The whole food intervention was so well accepted by the subjects that the blood lycopene (the primary carotenoid in tomatoes responsible for their red color) doubled and the prostate lycopene concentration tripled during this short period. Oxidative DNA damage in leukocytes and prostate tissues was significantly diminished, the latter mainly in the tumor cell nuclei, possibly due to the antioxidant properties of lycopene. Quite surprising was the decrease in blood prostate-specific antigen, which was explained by the increase in apoptotic death of prostate cells, especially in carcinoma regions. Prostate cancer cell cultures (LNCaP) were also sensitive to lycopene in growth medium, which caused an increased apoptosis and arrested the cell cycle. A possible explanation of these promising results may reside in lycopene effects on the genes governing the androgen stimulation of prostate growth, cytokines and on the enzymes producing reactive oxygen species, all of which were recently discovered by nutrigenomic techniques. Other phytochemicals in tomato may act in synergy with lycopene to potentiate protective effects and to help in the maintenance of prostate health.
Keywords: Carotenoid; Cancer; Oxidative stress; Apoptosis;

The effect of β-carotene and its derivatives on cytotoxicity, differentiation, proliferative potential and apoptosis on the three human acute leukemia cell lines: U-937, HL-60 and TF-1 by Tomasz Sacha; Magdalena Zawada; Jadwiga Hartwich; Zofia Lach; Anna Polus; Marta Szostek; Edyta Zdziƚowska; Marta Libura; Marek Bodzioch; Aldona Dembińska-Kieć; Aleksander B. Skotnicki; Regina Góralczyk; Karin Wertz; Georges Riss; Christopher Moele; Thomas Langmann; Gerd Schmitz (206-214).
The influence of β-carotene (BC) and its derivatives on differentiation, proliferation and apoptosis in three human acute leukemia cell lines was studied. We investigated: (i) the cellular uptake of BC, (ii) the cytotoxicity, (iii) the effect on cell cycle progression and/or apoptosis. The dose- and time-dependent pattern of cellular BC uptake in all studied cell lines was seen. We did not observe any cytotoxic effect of BC and ATRA in the chosen concentrations. There was only limited effect of BC on gene expression. The microarrray analysis of U-937 cell line exposed to BC for 72 h showed an increased expression of BAX gene. This finding was confirmed by real-time Q-PCR analysis, and supported by a flow cytometry apoptosis tests. We did not observe any influence of studied components on cellular proliferation. The induction of differentiation after incubation with ATRA in HL-60 cells was noted. The induction of cellular apoptosis by BC was seen in all studied cell lines. We demonstrated that BC used in the concentrations achievable in vivo does not affect the proliferation and differentiation process of the studied leukemic cell lines, but can influence and enhance the apoptosis by modulating the expression of the regulatory genes.
Keywords: β-Carotene; Carcinogenesis; Apoptosis; Human acute leukemia cell line;

Many studies suggest a protective role of β-carotene against cancer. However, the ATBC and the CARET trials have shown that β-carotene increases the incidence of lung cancer in heavy smokers and asbestos workers. To explain this paradox, it can be hypothesized that β-carotene modulates intracellular redox status and through this mechanism, it affects redox-sensitive molecular pathways involved in the regulation of cell cycle progression and apoptosis. Studies conducted in cultured cells seem to confirm such a hypothesis. At low concentrations, the carotenoid may serve as an antioxidant, inhibiting free radical production, while at relatively high concentrations and/or in the presence of a chronic oxidative stress (i.e. smoke), it may behave as a prooxidant, propagating free radical-induced reactions, consuming endogenous antioxidants and inducing DNA oxidative damage. In this context, it may regulate cell growth and death by the modulation of redox-sensitive genes and transcription factors.
Keywords: β-carotene; Intracellular fredox status; Cell growth;

Proangiogenic activity of beta-carotene is coupled with the activation of endothelial cell chemotaxis by A. Dembinska-Kiec; A. Polus; B. Kiec-Wilk; J. Grzybowska; M. Mikolajczyk; J. Hartwich; U. Razny; K. Szumilas; A. Banas; M. Bodzioch; J. Stachura; G. Dyduch; P. Laidler; J. Zagajewski; T. Langman; G. Schmitz (222-239).
Endothelial cells play an important role in angiogenesis (formation of new vessels from preexisting ones), which is essential for organogenesis, tissue remodeling but also inflammatory response, carcinogenesis in all periods of our life. Beta-carotene (BC) in non-toxic concentrations (up to 3 μM) had no detectable effect on HUVECs (human umbilical vein endothelial cells) proliferation or apoptosis, despite significant changes of the expression patterns of pro- and anti-apoptotic genes. However beta-carotene did not change the tubulogenic activity of HUVEC in the in vitro angiogenesis model, it potently accelerated the bFGF-induced development of microcapillaries, as well as the migration of endothelial cells, in matrigel plug injected subcutaneously to mice. Potent activation of endothelial cell migration in the in vitro model of chemotaxis was also observed. According to the microarray data, genes involved in cell/cell and cell/matrix adhesion, matrix reorganization, activation of chemotaxis, the G-protein regulated intracellular signaling as well as genes involved in the rapid remodeling of protein cytoskeleton were the most affected by BC in HUVEC. We conclude that beta-carotene in the physiological concentration range stimulates early steps of angiogenesis by the activation of cellular migration as well as matrix reorganization and decrease of cell adhesion.
Keywords: Beta-carotene; Angiogenesis; Endothelium; Microarray; Chemotaxis;

Embryonic stem cells for basic research and potential clinical applications in cardiology by Johannes Winkler; Jürgen Hescheler; Agapios Sachinidis (240-248).
Embryonic stem (ES) cells are pluripotent, possessing the unique property to differentiate into any somatic cell type while retaining the ability to proliferate indefinitely. Due to their ability to recapitulate embryonic differentiation, ES cells are an ideal tool to study the process of early embryogenesis in vitro. Signalling cascades and genes involved in differentiation can be easily studied, and functional genomics approaches aim to identify the regulatory networks underlying lineage commitment. Their unique ability to differentiate into any cell type make ES cells a prime candidate for cell replacement therapy (CRT) of various degenerative disorders. Results from various disease models are promising and have demonstrated their principal suitability as a therapeutic agent in diseases such as myocardial infarctions, diabetes mellitus and Parkinson's disease. Prior to clinical trials in humans, two issues remain to be solved: due to their high proliferative potential, ES cells can form teratocarcinomas in the recipient, and depending on the source of the cells, ES cell grafts may be rejected by the host organism. This review discusses the current state of basic ES cell research with a focus on cardiac differentiation and gives an overview of their use in CRT approaches.
Keywords: Embryonic stem cell; Cardiomyocyte; Cell replacement therapy; Signalling molecule; Growth factor; Graft rejection;

Retinoic acid modulates the retinoblastoma protein during adipocyte terminal differentiation by Joan Ribot; Paula Oliver; Francisca Serra; Andreu Palou (249-257).
Terminal differentiation is characterized by a permanent withdrawal of cells from the cell cycle. Retinoblastoma protein (RB) has been involved in cell cycle progression. Accumulating evidence also implicates RB in the promotion of differentiation of many cell types. We present new insights into the role of RB and other cell cycle regulatory proteins in adipocyte differentiation and on the role of retinoic acid (RA) in the regulation of the latter process. It is shown that RA reduces RB expression and enhances RB phosphorylation by a mechanism that involves down-regulation of the cyclin-dependent kinase inhibitor (CKI) p21Cip1, having this fact as important consequences for both the cell cycle progression and the adipocyte differentiation process. The effects of RA result in the blockage of adipogenesis, but may also favor the retention of a pool of adipose cells able to re-enter the cell cycle, which may be important for the developmental dynamics of adipose tissue in vivo. In addition, these results reinforce the idea that there is a cross-talk between the cell cycle machinery and the adipocyte differentiation machinery that can be modulated by external signals, including nutrients.
Keywords: Adipogenesis; Retinoic acid; Cell cycle; Retinoblastoma protein; p21Cip1;

Retinoic acid (RA) administration and chronic vitamin A supplementation were reported to inhibit adipose tissue leptin expression in rodents, but the impact of this effect on food intake and its relationship with changes of body adiposity was not analyzed. Here, we have studied the effects of RA administration at three different doses on body weight, adipose tissue mass, food intake, adipose tissue leptin expression and circulating leptin levels in NMRI mice; the effects of chronic vitamin A supplementation with a 40-fold excess retinyl palmitate on the same parameters in NMRI and C57BL/6J mice; and the effects of RA and retinoid receptors agonists on leptin expression in brown and white adipocyte cell model systems. The results show that vitamin A down-regulates leptin expression in white and brown adipose tissue and circulating leptin levels independently of changes of adipose tissue mass and, for the first time to our knowledge, that this effect does not correlate with increased food intake. They also demonstrate a direct inhibitory effect of RA on leptin expression in both white and brown adipocyte cell cultures, and constitute first proof of the involvement of both RA receptors (RARs) and rexinoid receptors (RXRs) in this effect. Reduction of leptin levels by specific nutrients is of potential interest from a clinical point of view.
Keywords: Leptin; Retinoic acid; Vitamin A supplementation; Food intake; RAR; RXR;

Regulation of adipocyte differentiation and function by polyunsaturated fatty acids by Lise Madsen; Rasmus Koefoed Petersen; Karsten Kristiansen (266-286).
A diet enriched in PUFAs, in particular of the n-3 family, decreases adipose tissue mass and suppresses development of obesity in rodents. Although several nuclear hormone receptors are identified as PUFA targets, the precise molecular mechanisms underlying the effects of PUFAs still remain to be elucidated. Here we review research aimed at elucidating molecular mechanisms governing the effects of PUFAs on the differentiation and function of white fat cells. This review focuses on dietary PUFAs as signaling molecules, with special emphasis on agonistic and antagonistic effects on transcription factors currently implicated as key players in adipocyte differentiation and function, including peroxisome proliferator activated receptors (PPARs) (alpha, beta and gamma), sterol regulatory element binding proteins (SREBPs) and liver X receptors (LXRs). We review evidence that dietary n-3 PUFAs decrease adipose tissue mass and suppress the development of obesity in rodents by targeting a set of key regulatory transcription factors involved in both adipogensis and lipid homeostasis in mature adipocytes. The same set of factors are targeted by PUFAs of the n-6 family, but the cellular/physiological responses are dependent on the experimental setting as n-6 PUFAs may exert either an anti- or a proadipogenic effect. Feeding status and hormonal background may therefore be of particular importance in determining the physiological effects of PUFAs of the n-6 family.
Keywords: Polyunsaturated fatty acid; Adipocyte differentiation; Peroxisome proliferator-activated receptor; Sterol regulatory element-binding protein; Liver X receptor;

Fatty acids and expression of adipokines by Christian A. Drevon (287-292).
Adipose tissue has been recognised as the quantitatively most important energy store of the human body for many years, in addition to its functions as mechanical and thermic insulator. In mammals, the adipose organ is localised in several depots including white as well as brown adipose tissues. The largest depots are found subcutaneously and in the abdominal region. Several secretory proteins are synthesised in adipose tissue including leptin, resistin, adiponectin, tumor necrosis factor (TNFα), angiotensinogen, adipsin, acylation-stimulating protein, retinol-binding protein (RBP), interleukin (IL)-1b, IL-6, IL-8, IL-10, plasminogen activator inhibitor-1 (PAI-1), fasting-induced adipose factor, fibrinogen-angiopoietin-related protein, metallothionein, tissue factor (TF), complement C3, fibronectin, haptoglobin, entactin/nidogen, collagen VI α 3, pigment epithelium-derived factor (PEDF), hippocampal cholinergic neurostimulating peptide (HCNP), neutrophil gelatinase-associated lipocalin (NGAL) and adiponutrin. Fatty acids may influence the expression of adipokines like leptin, resistin or adiponectin directly by interaction with transcription factors, or indirectly via unknown mechanisms possibly linked to fatty acid oxidation, synthesis or storage. Because fatty acids are the main components of adipose tissue, it is of essential interest to clarify the biological effects of different types of fatty acids on the expression of relevant adipokines.
Keywords: Fatty acid; Adipokine; Adipose tissue; Leptin; Resistin; Adiponectin;

PPARγ in the control of brown adipocyte differentiation by Jan Nedergaard; Natasa Petrovic; Eva M. Lindgren; Anders Jacobsson; Barbara Cannon (293-304).
The effects of fatty acids and retinoic acid (carotene) on brown adipose tissue differentiation are mediated by activation of the transcription factors PPARγ and PPARα in combination with RXR. There is good support for the idea that activated PPARγ promotes adipogenesis also in brown adipose tissue. However, the issue is more complex concerning the full differentiation to the brown adipocyte phenotype, particularly the expression of the brown-fat-specific marker UCP1. The effect of norepinephrine on PPARγ gene expression, at least in-vitro, is negative, PPARγ-ablated brown adipose tissue can express UCP1, and PGC-1α coactivates other transcription factors (including PPARα); thus, the significance of PPARγ for the physiological control of UCP1 gene expression is not settled. However, importantly, the effects of PPAR agonists demonstrate the existence of a pathway for brown adipose tissue recruitment that is not dependent on chronic adrenergic stimulation and may be active in recruitment conditions such as prenatal and prehibernation recruitment. The ability of chronic PPARγ agonist treatment to promote the occurrence of brown-fat features in white adipose tissue-like depots implies a role in anti-obesity treatment, but this will only be effective if the extra thermogenic capacity is activated by adrenergic stimulation.
Keywords: Brown adipose tissue; UCP1; PPARγ;

Morphology of ferret subcutaneous adipose tissue after 6-month daily supplementation with oral beta-carotene by Incoronata Murano; Manrico Morroni; Maria Cristina Zingaretti; Paula Oliver; Juana Sánchez; Antonia Fuster; Catalina Picó; Andreu Palou; Saverio Cinti (305-312).
Adipose tissue is an important retinoid depot and retinoids are known to influence white and brown adipocyte metabolism. Identifying nutrients that can affect the biological activity of the adipose organ would be of great medical interest in the light of the current obesity epidemic and related disorders in developed countries. The vast majority of mammal studies of chronic administration of oral beta-carotene have used murine models, while few have employed mammals exhibiting uptake and processing of intestinal beta-carotene similar to those of humans. While rodents transform practically all ingested beta-carotene into retinol, in ferrets, as in humans, part of the beta-carotene is absorbed and released into the circulation intact. We studied the effects of 6-month daily administration of two doses of oral beta-carotene (0.8 or 3.2 mg/kg/day) on ferret body weight, size of body fat depots, and, using morphological and morphometric methods, on subcutaneous (inguinal) white adipose tissue (WAT). Because of the oral mode of administration, liver, stomach, and small and large intestine were also studied. Control animals received the vehicle. Data show that at the end of treatment the higher dose induced significantly higher body weight compared with controls and significantly higher inguinal fat depot compared with animals treated with the lower dose. In addition, chronic treatment with beta-carotene induced a dose-dependent hypertrophy of white adipocytes and increased neoangiogenesis in subcutaneous WAT in all treated ferrets. Vasculogenesis was independent of adipocyte hypertrophy. We also found focally evident liver steatosis in the ferrets treated with the higher dose of beta-carotene. The other gastrointestinal tract organs studied were not significantly different from those of control animals.
Keywords: beta-carotene chronic administration; WAT morphology; Ferret;

Roles of PPAR delta in lipid absorption and metabolism: a new target for the treatment of type 2 diabetes by Serge Luquet; Celine Gaudel; Dorte Holst; Joaquin Lopez-Soriano; Chantal Jehl-Pietri; Alexandre Fredenrich; Paul A. Grimaldi (313-317).
Peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors exerting several functions in development and metabolism. PPARα, activated by polyunsaturated fatty acids and fibrates, is implicated in regulation of lipid metabolism, lipoprotein synthesis and metabolism and inflammatory response in liver and other tissues. PPARγ plays important roles in regulation of proliferation and differentiation of several cell types, including adipose cells. Its activation by thiazolidinediones results in insulin sensibilization and antidiabetic action. Until recently, the physiological functions of PPARδ remain elusive. The utilization of specific agonists and of appropriate cellular and animal models revealed that PPARδ has an important role in metabolic adaptation of several tissues to environmental changes. Treatment of obese animals by specific PPARδ agonists results in normalization of metabolic parameters and reduction of adiposity. The nuclear receptor appeared to be implicated in the regulation of fatty acid burning capacities of skeletal muscle and adipose tissue by controlling the expression of genes involved in fatty acid uptake, β-oxidation and energy uncoupling. PPARδ is also implicated in the adaptive metabolic response of skeletal muscle to endurance exercise by controlling the number of oxidative myofibers. Given the results obtained with animal models, PPARδ agonists may have therapeutic usefulness in metabolic syndrome by increasing fatty acid consumption in skeletal muscle and adipose tissue.
Keywords: PPAR; Fatty acid; Obesity; Type 2 diabetes; Metabolic syndrome;