Modulation of miRNA Expression by Dietary Polyphenols in apoE Deficient Mice: A New Mechanism of the Action of Polyphenols

Background: Polyphenols are the most abundant antioxidants in the human diet and are widespread constituents of fruits and beverages, such as tea, coffee or wine. Epidemiological, clinical and animal studies support a role of polyphenols in the prevention of various diseases, such as cardiovascular diseases, cancers or neurodegenerative diseases. Recent findings suggest that polyphenols could interact with cellular signaling cascades regulating the activity of transcription factors and consequently affecting the expression of genes. However, the impact of polyphenol on the expression of microRNA, small non-coding RNAs, has not yet been studied. The aim of this study was to investigate the impact of dietary supplementation with polyphenols at nutritional doses on miRNA expression in the livers of apolipoprotein E-deficient mice (apoE(-/-)) jointly with mRNA expression profiling. [br/] Methodology/Principal Findings: Using microarrays, we measured the global miRNA expression in the livers of wild-type (C57B6/J) mice or apoE(-/-) mice fed diets supplemented with one of nine different polyphenols or a control diet. This analysis revealed that knock-out of the apoE gene induced significant modulation in the expression of miRNA. Moreover, changes in miRNA expression were observed after polyphenol supplementation, and five miRNAs (mmu-miR-291b-5p, mmu-miR-296-5p, mmu-miR-30c-1*, mmu-miR-467b* and mmu-miR-374*) were identified as being commonly modulated by these polyphenols. We also observed that these polyphenols counteracted the modulation of miRNA expression induced by apoE mutation. Pathway analyses on these five miRNA-target genes revealed common pathways, some of which were also identified from a pathway analysis on mRNA profiles. [br/] Conclusion:This in vivo study demonstrated for the first time that polyphenols at nutritional doses modulate the expression of miRNA in the liver. Even if structurally different, all polyphenols induced a similar miRNA expression profile. Common pathways were identified from both miRNA-target and mRNA analysis, revealing cellular functions that could be regulated by polyphenols at both the miRNA and mRNA level

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 adipocyte

Effects of lycopene and ß-carotene on the adipose tissue physiology : a global positive impact on pathophysiological disorders associated to obesity ?

Le tissu adipeux est un organe complexe qui centralise de nombreuses fonctions métaboliques. La principale est la régulation de la balance énergétique. Depuis quelques années, il est reconnu pour être le siège d’une activité sécrétoire importante dont les produits sont les adipokines. Au cours du développement de l’obésité, il concentre de nombreux dysfonctionnements cellulaires qui vont avoir des répercutions fonctionnelles importantes, sur lui-même, mais également sur d’autres tissus. Ces dysfonctionnements vont êtres à l’origine de nombreuses complications liées à l’obésité dont l’insulino-résistance, le diabète de type II ou les maladies cardiovasculaires. Dans ce cadre, des études épidémiologiques ont montré que la consommation de fruits et légumes avait un impact bénéfique, attribué en partie à certains micronutriments dont les caroténoïdes, sur ces pathologies et sur certains types de cancer. Parmi ces caroténoïdes, le lycopène et le ß-carotène occupent une place importante. Ils représentent les deux principaux caroténoïdes de notre alimentation et sont stockés physiologiquement au niveau du tissu adipeux. Des études ont suggéré qu’ils étaient spécifiquement et individuellement liés à une diminution des pathologies cardiovasculaires et de ses complications. De plus, compte tenu du lien très fort existant entre tissu adipeux, obésité et les pathologies associées, des études ont suggéré qu’il existait une relation entre ces deux caroténoïdes, le tissu adipeux et ces pathologies. Cependant, à ce jour, il n’existe quasiment pas d’explications sur les liens entre ces trois facteurs. Le but de cette thèse est d’apporter les premières pistes explicatives. Nous avons ainsi évalué les effets du lycopène et du ß-carotène sur certains aspects de la biologie du tissu adipeux et des fonctions adipocytaires. Nous montrons que les deux isomères principaux du lycopène (all-trans et 5-cis), son métabolite et le ß-carotène influencent fortement le transcriptome et le microtranscriptome de l’adipocyte. De plus, le lycopène diminue la réponse inflammatoire en réponse à un régime riche en gras via une diminution de l’activité NF-?B, et que un de ses métabolites, l’acide apo-10’-lycopénoïque transactive RAR in vivo dans le tissu adipeux. Il possède également la même capacité antiinflammatoire. Le ß-carotène quant à lui diminue l’adiposité chez des souris. Ce mécanisme est BCMO1 et PPAR? dépendant. Enfin, L’ensemble de nos résultats apportent de nouvelles perspectives et élargissent les connaissances sur ces deux caroténoïdes. Ils sont capables d’influencer de façon importante de nombreuses fonctions adipocytaires en lien avec l’obésité et ses complications dont les maladies cardiovasculaires selon différents mécanismes.Adipose tissue is a complex organ who exerts several metabolic functions. It is involved in the regulation of the energy balance and since a decade is well known as an exocrine organ who secrets several proteins collectively called adipokines. It is one of the main organs involved in obesity where it concentrates several cellular dysfunctions which will have important physiological consequences such as development of insulin resistance, type II diabetes and cardiovascular diseases. Epidemiological studies have reported that high consumption of fruits and vegetables is link to a decrease of pathologies such as cardiovascular diseases, type II diabetes and cancer; due to the presence of micronutrients notably carotenoids. Among these carotenoids, lycopene and ß-carotene play an important role. They are the main carotenoids in our diet, and are physiologically store in adipose tissue. Furthermore, others studies point that the high consumption or concentration of these compounds in adipose tissue is associated to a decrease of the risk to develop cardiovascular diseases. However, the link between carotenoids, adipose tissue, obesity-associated pathologies is unclear. The aim of this thesis was to bring some explanatory way to this association. An analyze of the transcriptome and microtranscriptome of adipocytes in response to lycopene (all-trans and 5-cis, the two main isomers and the metabolite) and ß-carotene also reveal that these compounds influence a large amount of genes and miRNAs. These effects can also explain a part of the positives effects attributed to these carotenoids. We found that lycopene decrease the inflammatory state of adipose tissue in response to a high fat diet. This phenomenon is explained by a decrease of the NF-?B activity. We also found that a metabolite of lycopene, the apo-10’-lycopenoic acid is able to transactivate RAR in vivo in different tissues included adipose tissue and possess the same anti inflammatory property than all-trans lycopene. In another article, we show that, a diet rich in ß-carotene in mouse lead to a decrease of adiposity. This effect is BCMO1 and PPAR? dependant. This enzyme, BCMO1, seems to play a key role in adipose tissue. Taking all these results together, we open new ways related to the effect of lycopene and ß- carotene on adipose tissue. These results can explain at least in part the benefic effects observed in several studies

Effets du lycopène et du ß-carotène sur la physiologie du tissu adipeux : un impact globalement positif sur les désordres physiopathologiques associés à l'obésité ?

Adipose tissue is a complex organ who exerts several metabolic functions. It is involved in the regulation of the energy balance and since a decade is well known as an exocrine organ who secrets several proteins collectively called adipokines. It is one of the main organs involved in obesity where it concentrates several cellular dysfunctions which will have important physiological consequences such as development of insulin resistance, type II diabetes and cardiovascular diseases. Epidemiological studies have reported that high consumption of fruits and vegetables is link to a decrease of pathologies such as cardiovascular diseases, type II diabetes and cancer; due to the presence of micronutrients notably carotenoids. Among these carotenoids, lycopene and ß-carotene play an important role. They are the main carotenoids in our diet, and are physiologically store in adipose tissue. Furthermore, others studies point that the high consumption or concentration of these compounds in adipose tissue is associated to a decrease of the risk to develop cardiovascular diseases. However, the link between carotenoids, adipose tissue, obesity-associated pathologies is unclear. The aim of this thesis was to bring some explanatory way to this association. An analyze of the transcriptome and microtranscriptome of adipocytes in response to lycopene (all-trans and 5-cis, the two main isomers and the metabolite) and ß-carotene also reveal that these compounds influence a large amount of genes and miRNAs. These effects can also explain a part of the positives effects attributed to these carotenoids. We found that lycopene decrease the inflammatory state of adipose tissue in response to a high fat diet. This phenomenon is explained by a decrease of the NF-?B activity. We also found that a metabolite of lycopene, the apo-10’-lycopenoic acid is able to transactivate RAR in vivo in different tissues included adipose tissue and possess the same anti inflammatory property than all-trans lycopene. In another article, we show that, a diet rich in ß-carotene in mouse lead to a decrease of adiposity. This effect is BCMO1 and PPAR? dependant. This enzyme, BCMO1, seems to play a key role in adipose tissue. Taking all these results together, we open new ways related to the effect of lycopene and ß- carotene on adipose tissue. These results can explain at least in part the benefic effects observed in several studies.Le tissu adipeux est un organe complexe qui centralise de nombreuses fonctions métaboliques. La principale est la régulation de la balance énergétique. Depuis quelques années, il est reconnu pour être le siège d’une activité sécrétoire importante dont les produits sont les adipokines. Au cours du développement de l’obésité, il concentre de nombreux dysfonctionnements cellulaires qui vont avoir des répercutions fonctionnelles importantes, sur lui-même, mais également sur d’autres tissus. Ces dysfonctionnements vont êtres à l’origine de nombreuses complications liées à l’obésité dont l’insulino-résistance, le diabète de type II ou les maladies cardiovasculaires. Dans ce cadre, des études épidémiologiques ont montré que la consommation de fruits et légumes avait un impact bénéfique, attribué en partie à certains micronutriments dont les caroténoïdes, sur ces pathologies et sur certains types de cancer. Parmi ces caroténoïdes, le lycopène et le ß-carotène occupent une place importante. Ils représentent les deux principaux caroténoïdes de notre alimentation et sont stockés physiologiquement au niveau du tissu adipeux. Des études ont suggéré qu’ils étaient spécifiquement et individuellement liés à une diminution des pathologies cardiovasculaires et de ses complications. De plus, compte tenu du lien très fort existant entre tissu adipeux, obésité et les pathologies associées, des études ont suggéré qu’il existait une relation entre ces deux caroténoïdes, le tissu adipeux et ces pathologies. Cependant, à ce jour, il n’existe quasiment pas d’explications sur les liens entre ces trois facteurs. Le but de cette thèse est d’apporter les premières pistes explicatives. Nous avons ainsi évalué les effets du lycopène et du ß-carotène sur certains aspects de la biologie du tissu adipeux et des fonctions adipocytaires. Nous montrons que les deux isomères principaux du lycopène (all-trans et 5-cis), son métabolite et le ß-carotène influencent fortement le transcriptome et le microtranscriptome de l’adipocyte. De plus, le lycopène diminue la réponse inflammatoire en réponse à un régime riche en gras via une diminution de l’activité NF-?B, et que un de ses métabolites, l’acide apo-10’-lycopénoïque transactive RAR in vivo dans le tissu adipeux. Il possède également la même capacité antiinflammatoire. Le ß-carotène quant à lui diminue l’adiposité chez des souris. Ce mécanisme est BCMO1 et PPAR? dépendant. Enfin, L’ensemble de nos résultats apportent de nouvelles perspectives et élargissent les connaissances sur ces deux caroténoïdes. Ils sont capables d’influencer de façon importante de nombreuses fonctions adipocytaires en lien avec l’obésité et ses complications dont les maladies cardiovasculaires selon différents mécanismes

PPARβ promotes differentiation but represses dorsal mesoderm and endoderm specification.

<p>(A)–(C) Embryos were injected with PPARβ MO or Co, allowed to develop until stage 18 (A) or stage 11.5 (B) and (D), and collected for extraction of total RNA. qRT-PCR runs for a selection of neural (blue), mesodermal (red), or endodermal (yellow) markers of differentiation (A) and (B) or of germ layer specification (C) were conducted. RNA levels were normalized to EEF1a and RPL8 and are presented as fold variation between MO and Co samples. Error bars represent the S.E.M. of 3 to 5 independent experiments. (D) Embryos were injected with PPARβ MO or Co, fixed at stg. 11.5, hemi-sectioned along the dorso–ventral axis, and processed for RNA <i>in situ</i> hybridization. While Mo injection did not affect the <i>sox17α</i> expression domain, it resulted in the expansion of <i>brachyury</i> expression dorsally (see the scale) but not ventrally. Arrows indicate the dorsal lip. (E) Quantification of the surface covered by the dorsal and ventral expression domains of <i>brachyury</i> in MO compared to Co hemi-sections. Error bar is the S.E.M. of 10 measurements. *: two-tailed Student’s t-test vs control, P<0.05.</p

Rate of transcript level variation is maximal at gastrula stage.

<p>(A) Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083300#pone.0083300-Irie1" target="_blank">[23]</a> were used to quantify transcription variations during normal development. The number of genes showing an RNA level increase or decrease by 2×, 4×, or 8× between two consecutive stages was plotted. Data were normalized by the duration, in hours, of each developmental period analysed. (B) The group of genes with RNA levels that increased 4× or more between stage 11 and stage 13 was considered, and the RNA levels of these genes were plotted at different developmental stages. The rectangles delineate the 25^th and 75^th percentiles, the horizontal bar is the median, and the whiskers indicate the 10^th and 90^th percentiles.</p

PPARβ promotes the initiation of differentiation at gastrulation.

<p>(A) Rationale of the transcriptomic analysis of PPARβ loss-of-function. (B) The gene set consisting of predicted direct PPAR target genes in humans <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083300#pone.0083300-Heinaniemi1" target="_blank">[33]</a> was analysed by GSEA. (C) The Gene Ontology terms or the gene sets that were significantly (FDR<0.2) affected by PPARβ loss-of-function are presented. The gene sets corresponding to germ layer specification are also presented. (D) The gene sets consisting of the 100 most-induced genes and of the 100 most-decreased genes at gastrula (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083300#pone.0083300.s005" target="_blank">Fig. S5</a>) were analysed by GSEA. FDR, false discovery rate; GSEA, Gene Set Enrichment Analysis; NES, Normalized enrichment score.</p

PPARβ is essential for <i>X. laevis</i> development.

<p>(A) Design of the morpholino (PPARβ MO) to target PPARβ translation and of the control morpholino (Co). Capital letters designate nucleotides that can hybridize with the PPARβ MO. (B) Immunoblot showing endogenous PPARβ levels in non-injected embryos (Ni) and embryos injected with PPARβ MO or Co. β-actin served as a loading control. (C) Scoring of A–P axis defects. Different doses of PPARβ MO, Co, or a combination of PPARβ MO and PPARβ_rescue mRNA were injected. Embryos with a length about a third of that of non-injected sibling embryos were scored as ‘very-short axis’, and those with a length of about two thirds of normal were scored as ‘short axis’. (D) Representative not-injected (Ni), Co-injected (Co), MO-injected (MO), and MO combined with rescue injected (PPARβ MO + PPARβ_rescue) embryos.</p

PPARβ interprets a chromatin signature that is deposited at the end of the pluripotent stage.

<p>(A) Seven ‘K27’ genes and eight ‘K4 only’ genes were analysed by ChIP as indicated. Results are presented in a heat map (see also Supplementary Fig. 7). Variation in RNA expression upon PPARβ MO injection was obtained from the RNA-seq data or from qPCR validations. (B) ChIP with H3K27me3 antibody was conducted at stage 9 on 37 ‘PPARβ promoted genes’ and on 27 Control genes. PPARβ promoted genes were chosen among the top 200 most downregulated genes at stage 11, upon MO injection in the list presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083300#pone.0083300.s008" target="_blank">Table S1</a>, while Control genes did not show a change of expression upon MO injection. Results are presented as percentage of input. The threshold of 1% is indicated. Genes scored as positive for H3K27me3 are indicated by a red dot (see methods for further details on the definition of gene sets and on the criteria of scoring). (C) Sequential ChIPs were conducted. Note that no enrichment was observed for klf11 and for plcg1, which represent negative controls (see panel b). Error is the S.E.M of 2 independent experiments. (D) ChIP using PPARβ antibody was conducted at stage 11.5. Error is the S.E.M of 3 to 4 independent experiments. (E) ChIP with H3K27me3 antibody or PPARβ antibody and <i>q</i>RT-PCR were conducted on embryos treated with DZNep or DMSO and injected with PPARβ MO or Co. Error is the S.E.M of technical replicates of a single experiment that we have replicated with similar results.</p

Purified low-density lipoprotein and bovine serum albumin efficiency to internalise lycopene into adipocytes

International audienceEpidemiological studies have suggested that lycopene has protective effects against various diseases including cardiovascular diseases. However, mechanistic studies to understand these effects are difficult due to the insolubility of lycopene in aqueous culture medium. The objective of the present study was to use LDL or BSA as physiological vehicles for lycopene and to compare them with various classical vehicles. Among tested vehicles, only LDL, BSA, THF/BHT, beadlets, and liposomes were able to solubilise lycopene. No cytotoxicity was observed with these vehicles. LDL and BSA allowed good stability of lycopene during incubation (52% and 43% for 2microM lycopene solutions), but remained less efficient than THF/BHT or beadlets (67% and 62%). Incubation of adipocytes (3T3-L1) with the different vehicles for 24 and 48h showed that beadlets best delivered lycopene to cells. Finally, whatever the vehicle used, intracellular localization of lycopene was the same: lipid droplets (32-51%), plasma membrane (32-37%) and nuclear membrane (19-29%). As a conclusion, LDL or BSA display comparable properties to THF/BHT or beadlets. It is the first time that lycopene carried by physiological vehicles is shown to reach different subcellular compartments supporting molecular effects in adipocyte, such as cell signaling or nuclear receptor interacting