Jean-François Landrier

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. 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. 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.

Lycopene inhibits proinflammatory cytokine and chemokine expression in adipose tissue

Obesity is associated with a low-grade inflammation which is correlated with an increased secretion of pro-inflammatory cytokines and chemokines by adipose tissue, suspected to contribute to the development of insulin resistance. Because lycopene is mostly stored in adipose tissue and possesses anti-inflammatory properties, we hypothesize that lycopene could reduce the production of proinflammatory markers in adipose tissue. In agreement with this hypothesis, we observed a decrease of inflammatory markers such as IL-6, MCP-1 and IL-1β at both the mRNA and protein level when explants of epididymal adipose tissue from mice fed with a high-fat diet were incubated with lycopene ex vivo. The same effect was reproduced with explants of adipose tissue preincubated in lycopene and then subjected to TNFα stimulation. The contribution of adipocytes and preadipocytes was evaluated. In both preadipocytes and differentiated 3T3-L1 adipocytes, lycopene preincubation for 24 h decreased the TNFα-mediated induction of IL-6 and MCP-1. Finally, the same results were reproduced with human adipocyte primary cultures. The molecular mechanism was also studied. In transient transfections, a decrease of the luciferase gene reporter under control of NF-κB responsive element was observed for cells incubated in the presence of lycopene and TNFα compared to TNFα alone. The involvement of the NF-κB pathway was confirmed by the modulation of IKKα/β phosphorylation by lycopene. Altogether, these results showed for the first time a limiting effect of lycopene on adipose tissue proinflammatory cytokine and chemokine production. Such an effect could prevent or limit the prevalence of obesity-associated pathologies, such as insulin resistance.

Vitamin D intestinal absorption is not a simple passive diffusion: Evidences for involvement of cholesterol transporters

SCOPE It is assumed that vitamin D is absorbed by passive diffusion. However, since cholecalciferol (vitamin D(3) ) and cholesterol display similar structures, we hypothesized that common absorption pathways may exist. METHODS AND RESULTS Cholecalciferol apical transport was first examined in human Caco-2 and transfected Human embryonic kidney (HEK) cells. Cholecalciferol uptake was then valuated ex vivo and in vivo, using either wild-type mice, mice overexpressing Scavenger Receptor class B type I (SR-BI) at the intestinal level or mice treated or not with ezetimibe. Cholecalciferol uptake was concentration-, temperature- and direction-dependent, and was significantly impaired by a co-incubation with cholesterol or tocopherol in Caco-2 cells. Moreover Block Lipid Transport-1 (SR-BI inhibitor) and ezetimibe glucuronide (Niemann-Pick C1 Like 1 inhibitor) significantly decreased cholecalciferol transport. Transfection of HEK cells with SR-BI, Cluster Determinant 36 and Niemann-Pick C1 Like 1 significantly enhanced vitamin D uptake, which was significantly decreased by the addition of Block Lipid Transport-1, sulfo-N-succinimidyl oleate (Cluster Determinant 36 inhibitor) or ezetimibe glucuronide, respectively. Similar results were obtained in mouse intestinal explants. In vivo, cholecalciferol uptake in proximal intestinal fragments was 60% higher in mice overexpressing SR-BI than in wild-type mice (p<0.05), while ezetimibe effect remained non-significant. CONCLUSION These data show for the first time that vitamin D intestinal absorption is not passive only but involves, at least partly, some cholesterol transporters.

Vitamin D reduces the inflammatory response and restores glucose uptake in adipocytes.

SCOPE Obesity is strongly associated with low-grade inflammation, notably due to an overproduction of proinflammatory markers by adipose tissue and adipocytes as well as a vitamin D deficiency. Whether these problems are interrelated has not been clearly established. METHODS AND RESULTS In the present report, decreases in the levels of inflammatory markers such as IL-6, MCP-1, and IL-1β (mRNA and protein level) in human adipocytes and in 3T3-L1 adipocytes were observed after 1,25-dihydroxyvitamin D3 (1,25-(OH)(2) D(3) ) treatment. Such treatment also decreased the expression of the TNF-α-mediated proinflammatory marker in 3T3-L1 and human adipocytes. A similar effect was observed in adipocyte-macrophage co-culture systems in which 1,25-(OH)(2) D(3) decreased proinflammatory marker expression under basal and TNF-α-stimulated conditions. The involvement of VDR and NF-κB was confirmed in these regulations. Incubation with 1,25-(OH)(2) D(3) also resulted in the dephosphorylation of p38, which is linked to the transcriptional induction of several Dusp family members. Functional consequences of the 1,25-(OH)(2) D(3) treatment on glucose uptake and AKT phosphorylation were observed. CONCLUSION The improvement of both proinflammatory status and glucose uptake in adipocytes under 1,25-(OH)(2) D(3) effect suggests that low-grade inflammation could be linked to vitamin D deficiency. This observation offers new perspectives in the context of obesity and associated physiopathological disorders.

Lycopene attenuates LPS-induced TNF-α secretion in macrophages and inflammatory markers in adipocytes exposed to macrophage-conditioned media

SCOPE Adipose tissue is infiltrated by an increasing number of macrophages during the development of obesity. These immune cells are suspected to be a major source of TNF-α that interferes with adipocyte function. Because lycopene possesses anti-inflammatory properties, we hypothesize that lycopene could reduce the production of TNF-α by macrophages and thus interfere in the cross-talk between macrophages and adipocytes. METHODS AND RESULTS We demonstrated that physiological concentrations of lycopene were able to attenuate the lipopolysaccharide (LPS)-mediated induction of TNF-α in RAW 264.7 macrophages, at both the mRNA and protein levels. The molecular mechanism was studied. It appeared that the LPS-activation of both JNK and NF-κB signaling pathways was modulated by lycopene. The anti-inflammatory effects of lycopene on macrophages were accompanied by a decrease in LPS-stimulated macrophage migration in the presence of lycopene. Furthermore, lycopene decreased macrophage conditioned medium-induced proinflammatory cytokine, acute phase protein, and chemokine mRNA expression in 3T3-L1 adipocytes. CONCLUSION These data indicate that lycopene displayed an anti-inflammatory effect on macrophages that beneficially impacted adipocyte function. Thus, these results suggest that lycopene could block the vicious cycle that occurs between adipocytes and macrophages in adipose tissue during obesity.

CD36 is involved in lycopene and lutein uptake by adipocytes and adipose tissue cultures

SCOPE Carotenoids are mainly stored in adipose tissue. However, nothing is known regarding the uptake of carotenoids by adipocytes. Thus, our study explored the mechanism by which lycopene and lutein, two major human plasma carotenoids, are transported. METHODS AND RESULTS CD36 was a putative candidate for this uptake, 3T3-L1 cells were treated with sulfosuccinimidyl oleate, a CD36-specific inhibitor. sulfosuccinimidyl oleate-treated cells showed a significant decrease in both lycopene and lutein uptake as compared to control cells. Their uptake was also decreased by partial inhibition of CD36 expression using siRNA, whereas the overexpression of CD36 in Cos-1 cells increased their uptake. Finally, the effect of CD36 on carotenoid uptake was confirmed ex vivo in cultures of adipose tissue explants from CD36(-/-) mice, which exhibited reduced carotenoid uptake as compared to wild-type mice explants. CONCLUSION For the first time, we report the involvement of a transporter, CD36, in carotenoid uptake by adipocytes and adipose tissue.

Vitamin D protects against diet-induced obesity by enhancing fatty acid oxidation

Prospective studies reported an inverse correlation between 25-hydroxyvitamin D [25(OH)D] plasma levels and prevalence of obesity and type 2 diabetes. In addition, 25(OH)D status may be a determinant of obesity onset. However, the causality between these observations is not yet established. We studied the preventive effect of vitamin D3 (VD3) supplementation (15,000 IU/kg of food for 10 weeks) on onset of obesity in a diet-induced obesity mouse model. We showed that the VD3 supplementation limited weight gain induced by high-fat diet, which paralleled with an improvement of glucose homeostasis. The limitation of weight gain could further be explained by an increased lipid oxidation, possibly due to an up-regulation of genes involved in fatty acid oxidation and mitochondrial metabolism, leading to increased energy expenditure. Altogether, these data show that VD3 regulates energy expenditure and suggest that VD3 supplementation may represent a strategy of preventive nutrition to fight the onset of obesity and associated metabolic disorders.

Muscle ectopic fat deposition contributes to anabolic resistance in obese sarcopenic old rats through eIF2α activation.

Obesity and aging are characterized by decreased insulin sensitivity (IS) and muscle protein synthesis. Intramuscular ceramide accumulation has been implicated in insulin resistance during obesity. We aimed to measure IS, muscle ceramide level, protein synthesis, and activation of intracellular signaling pathways involved in translation initiation in male Wistar young (YR, 6‐month) and old (OR, 25‐month) rats receiving a low‐ (LFD) or a high‐fat diet (HFD) for 10 weeks. A corresponding cellular approach using C2C12 myotubes treated with palmitate to induce intracellular ceramide deposition was taken. A decreased ability of adipose tissue to store lipids together with a reduced adipocyte diameter and a development of fibrosis were observed in OR after the HFD. Consequently, OR fed the HFD were insulin resistant, showed a strong increase in intramuscular ceramide level and a decrease in muscle protein synthesis associated with increased eIF2α phosphorylation. The accumulation of intramuscular lipids placed a lipid burden on mitochondria and created a disconnect between metabolic and regulating pathways in skeletal muscles of OR. In C2C12 cells, palmitate‐induced ceramide accumulation was associated with a decreased protein synthesis together with upregulated eIF2α phosphorylation. In conclusion, a reduced ability to expand adipose tissues was found in OR, reflecting a lower lipid buffering capacity. Muscle mitochondrial activity was affected in OR conferring a reduced ability to oxidize fatty acids entering the muscle cell. Hence, OR were more prone to ectopic muscle lipid accumulation than YR, leading to decreased muscle protein anabolism. This metabolic change is a potential therapeutic target to counter sarcopenic obesity.

Vitamin E decreases endogenous cholesterol synthesis and apo-AI-mediated cholesterol secretion in Caco-2 cells☆

Intestine is the gateway for newly absorbed tocopherols. This organ also plays a crucial role in cholesterol metabolism. Because tocopherols are known to impact cholesterol metabolism in the liver, we hypothesized that tocopherols could also modulate cholesterol metabolism in the intestine. This study aimed to verify this hypothesis and to unveil the mechanisms involved, using Caco-2 cells as a model of the human intestinal cell. Both α- and γ-tocopherol significantly (P<.05) decreased endogenous cholesterol synthesis and apo-AI-mediated cholesterol secretion in Caco-2 cells. Tocopherols down-regulated (P<.05) up to half of the genes involved in the cholesterol synthesis pathway, together with CYP27A1, which is involved in oxysterol production. The activity of this enzyme, as well as the levels of intracellular oxysterols, was significantly diminished by tocopherols. Finally, tocopherols significantly reduced ABCA1 mRNA levels in Caco-2 cells. We conclude that tocopherols impair the endogenous synthesis and apo-AI-mediated secretion of cholesterol in Caco-2 cells. This effect involves a down-regulation of genes involved in the cholesterol synthesis pathway, resulting in down-regulation of CYP27A1 which, in turn, diminishes oxysterol concentrations. The outcome is a decrease of LXR activity, resulting in down-regulation of ABCA1. These data reinforce the effect of α- and γ-tocopherol on cholesterol metabolism via gene expression regulation.

Chemokine Expression in Inflamed Adipose Tissue Is Mainly Mediated by NF-κB

Immune cell infiltration of expanding adipose tissue during obesity and its role in insulin resistance has been described and involves chemokines. However, studies so far have focused on a single chemokine or its receptor (especially CCL2 and CCL5) whereas redundant functions of chemokines have been described. The objective of this work was to explore the expression of chemokines in inflamed adipose tissue in obesity. Human and mouse adipocytes were analyzed for expression of chemokines in response to inflammatory signal (TNF-α) using microarrays and gene set enrichment analysis. Gene expression was verified by qRT-PCR. Chemokine protein was determined in culture medium with ELISA. Chemokine expression was investigated in human subcutaneous adipose tissue biopsies and mechanism of chemokine expression was investigated using chemical inhibitors and cellular and animal transgenic models. Chemokine encoding genes were the most responsive genes in TNF-α treated human and mouse adipocytes. mRNA and protein of 34 chemokine genes were induced in a dose-dependent manner in the culture system. Furthermore, expression of those chemokines was elevated in human obese adipose tissue. Finally, chemokine expression was reduced by NF-κB inactivation and elevated by NF-κB activation. Our data indicate that besides CCL2 and CCL5, numerous other chemokines such as CCL19 are expressed by adipocytes under obesity-associated chronic inflammation. Their expression is regulated predominantly by NF-κB. Those chemokines could be involved in the initiation of infiltration of leukocytes into obese adipose tissue.