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Aim: Epidemiological evidence suggests adherence to vegetable-rich diets is associated to atheroprotective effects and bioactive components are most likely to play a relevant role. The notion of inter-kingdom regulation has opened a new research paradigm and perhaps microRNAs (miRNAs) from edible vegetables could influence consumer gene expression and lead to biological effects. We aimed to investigate the potential impact of broccoli-derived miRNAs on cellular cholesterol efflux in vitro., Methods: Four miRNAs (miR159a, miR159b, miR166a and miR403) from Brassica oleracea var. italica (broccoli), a widely consumed cruciferous vegetable, were selected for further investigation, based on their high abundancy in this vegetable and their presence in other plants. Selected miRNAs were synthesized with a 3'-terminal 2'-O-methylation and their cellular toxicity, in vitro gastrointestinal resistance and cellular uptake were evaluated. Potential target genes within the mammalian transcriptome were assessed in silico following pathway analysis. In vitro cholesterol efflux was assessed in human THP-1-derived macrophages., Results: miRNAs survival to in vitro GI digestion was around 1%, although some variation was seen between the four candidates. Cellular uptake by mammalian cells was confirmed, and an increase in cholesterol efflux was observed. Pathway analysis suggested these miRNAs are involved in biological processes related to phosphorylation, phosphatidylinositol and Wnt signaling, and to the insulin/IGF pathway., Conclusions: Health-promoting properties attributed to cruciferous vegetables, might be mediated (at least in part) through miRNA-related mechanisms., (Copyright © 2024 Sociedad Española de Arteriosclerosis. Publicado por Elsevier España, S.L.U. All rights reserved.)

Scope: Extra virgin olive oil has numerous cardiopreventive effects, largely due to its high content of (poly)phenols such as hydroxytyrosol (HT). However, some animal studies suggest that its excessive consumption may alter systemic lipoprotein metabolism. Because human lipoprotein metabolism differs from that of rodents, this study examines the effects of HT in a humanized mouse model that approximates human lipoprotein metabolism., Methods and Results: Mice are treated as follows: control diet or diet enriched with HT. Serum lipids and lipoproteins are determined after 4 and 8 weeks. We also analyzed the regulation of various genes and miRNA by HT, using microarrays and bioinformatic analysis. An increase in body weight is found after supplementation with HT, although food intake was similar in both groups. In addition, HT induced the accumulation of triacylglycerols but not cholesterol in different tissues. Systemic dyslipidemia after HT supplementation and impaired glucose metabolism are observed. Finally, HT modulates the expression of genes related to lipid metabolism, such as Pltp or Lpl., Conclusion: HT supplementation induces systemic dyslipidemia and impaired glucose metabolism in humanized mice. Although the numerous health-promoting effects of HT far outweigh these potential adverse effects, further carefully conducted studies are needed., (© 2023 Wiley-VCH GmbH.)

Cell-to-cell communication strategies include extracellular vesicles (EVs) in plants and animals. The bioactive molecules in a diet rich in vegetables and fruits are associated with disease-preventive effects. Plant-derived EVs (PDEVs) are biogenetically and morphologically comparable to mammalian EVs and transport bioactive molecules, including miRNAs. However, the biological functions of PDEVs are not fully understood, and standard isolation protocols are lacking. Here, PDEVs were isolated from four foods with a combination of ultracentrifugation and size exclusion chromatography, and evaluated as vehicles for enhanced transport of synthetic miRNAs. In addition, the role of food-derived EVs as carriers of dietary (poly)phenols and other secondary metabolites was investigated. EVs from broccoli, pomegranate, apple, and orange were efficiently isolated and characterized. In all four sources, 4 miRNA families were present in tissues and EVs. miRNAs present in broccoli and fruit-derived EVs showed a reduced RNase degradation and were ferried inside exposed cells. EVs transfected with a combination of ath-miR159a, ath-miR162a-3p, ath-miR166b-3p, and ath-miR396b-5p showed toxic effects on human cells, as did natural broccoli EVs alone. PDEVs transport trace amounts of phytochemicals, including flavonoids, anthocyanidins, phenolic acids, or glucosinolates. Thus, PDEVs can act as nanocarriers for functional miRNAs that could be used in RNA-based therapy., Competing Interests: Declaration of Competing Interest none. The authors declare no conflict of interest associated with this work., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose of Review: Accumulating data on the consumption of plant-based diets and their impact on blood pressure indicate a consensus that plant-based diets are linked to reduced blood pressure. The suggested mechanisms of action are manifold, and, in this systematic review, we provide a summary of the most recent findings on plant-based diets and their impact on blood pressure, along with an analysis of the molecules accountable for the observed effects., Recent Findings: The overwhelming majority of intervention studies demonstrate that plant-based diets result in lower blood pressure readings when compared to diets that are based on animal products. The various mechanisms of action are being clarified. The data discussed in this systematic review allow us to conclude that plant-based diets are associated with lower blood pressure and overall better health outcomes (namely, on the cardiovascular system) when compared to animal-based diets. The mechanisms of action are being actively investigated and involve many macro- and micronutrients plentiful in plants and the dishes prepared with them., (© 2023. The Author(s).)

Mitochondrial epitranscriptomics refers to the modifications occurring in all the different RNA types of mitochondria. Although the number of mitochondrial RNA modifications is less than those in cytoplasm, substantial evidence indicates that they play a critical role in accurate protein synthesis. Recent evidence supported those modifications in mitochondrial RNAs also have crucial implications in mitochondrial-related diseases. In the light of current knowledge about the involvement, the association between mitochondrial RNA modifications and diseases arises from studies focusing on mutations in both mitochondrial and nuclear DNA genes encoding enzymes involved in such modifications. Here, we review the current evidence available for mitochondrial RNA modifications and their role in metabolic disorders, and we also explore the possibility of using them as promising targets for prevention and early detection. Finally, we discuss future directions of mitochondrial epitranscriptomics in these metabolic alterations, and how these RNA modifications may offer a new diagnostic and theragnostic avenue for preventive purposes. This article is categorized under: RNA Processing > RNA Editing and Modification., (© 2022 The Authors. WIREs RNA published by Wiley Periodicals LLC.)

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression. The wide-ranging biological activities of microRNAs stimulated research on disease mechanisms and is suggesting appealing therapeutic applications. When unprotected, miRNAs suffer from rapid degradation and appropriate strategies need to be developed to improve their therapeutic potential. Since the first observation of miRNAs being naturally transported by extracellular vesicles (EVs), the latter have been proposed as specific transport means for drug delivery, conferring stability and increasing resistance against RNase degradation. However, a standard, reproducible, and cost-effective protocol for EV isolation is lacking. Here, the use of broccoli-derived EVs as a therapeutic vehicle for extracellular RNA drug delivery was assessed. EVs were isolated from broccoli, combining ultracentrifugation and size exclusion chromatography methodology. Caco-2 cells were exposed to isolated EVs loaded with exogenous miRNAs and cellular viability was tested. The miRNAs were taken up by this intestinal cell line. Our results show that broccoli EVs can be efficiently isolated, characterized, and loaded with exogenous miRNAs, leading to toxicity in caco-2 cells. Because the pharmaceutical industry is searching for novel drug delivery nanovesicles with intrinsic properties such as low immunogenicity, stability to the gastrointestinal tract, ability to overcome biological barriers, large-scale production, cost-effectiveness, etc., broccoli-isolated nanovesicles might be suitable candidates for future pharmacological applications. We propose broccoli as a natural source of EVs, which are capable of transporting exogenous miRNAs with potential therapeutic effects and suggest that appropriate toxicological and randomized controlled trials as well as patent applications are warranted., Competing Interests: Declaration of interest The authors declare no conflict of interest associated with this work., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Atherosclerosis is a hallmark of cardiovascular disease, and lifestyle strongly impacts its onset and progression. Nutrients have been shown to regulate the miR-17/92 cluster, with a role in endothelial function and atherosclerosis. Choline, betaine, and L-carnitine, found in animal foods, are metabolized into trimethylamine (TMA) by the gut microbiota. TMA is then oxidized to TMAO, which has been associated with atherosclerosis. Our aim was to investigate whether TMAO modulates the expression of the miR-17/92 cluster, along with the impact of this modulation on the expression of target genes related to atherosclerosis and inflammation. We treated HepG-2 cells, THP-1 cells, murine liver organoids, and human peripheral mononuclear cells with 6 µM of TMAO at different timepoints. TMAO increased the expression of all analyzed members of the cluster, except for miR-20a-5p in murine liver organoids and primary human macrophages. Genes and protein levels of SERPINE1 and IL-12A increased. Both have been associated with atherosclerosis and cardiovascular disease (CDVD) and are indirectly modulated by the miR-17-92 cluster. We concluded that TMAO modulates the expression of the miR-17/92 cluster and that such modulation could promote inflammation through IL-12A and blood clotting through SERPINE1 expression, which could ultimately promote atherosclerosis and CVD.

Purpose: Exosomes are extracellular vesicles secreted by cells, which can transport different molecules, including nucleic acids. Dietary habits may induce gene regulation through the modulation of exosomal RNAs. We aimed at characterizing exosomal lncRNAs, mRNA and miRNAs modulation after a 1-year adherence to a low-fat diet (LFD) or to Mediterranean-based diets enriched in extra-virgin olive oil (MedDiet + EVOO) or in a mixture of nuts (MedDiet + Nuts)., Methods: Plasma samples were collected, at baseline and after 1 year of dietary interventions, from 150 participants included in the PREDIMED study (Reus Center). LncRNAs, mRNAs and miRNAs were isolated from plasma exosomes and screened. RT-qPCR validation was performed for miRNAs., Results: Compared with LFD, 413 lncRNAs and 188 mRNAs, and 476 lncRNAs and 235 mRNAs were differentially modulated in response to the MedDiet + EVOO and MedDiet + Nuts interventions, respectively. In addition, after 1 year of dietary interventions, 26 circulating miRNAs were identified as differentially expressed between groups. After 1 year of intervention, 11 miRNAs significantly changed in LFD group, while 8 and 21 were modulated in response to the MedDiet enriched with EVOO or nuts, respectively. Bioinformatic analyses of differentially expressed miRNAs and their validated target genes suggest certain metabolic pathways are modulated by LFD (PI3K-Akt and AMPK), MedDiet + EVOO (PI3K-Akt, NF-kappa B, HIF-1, and insulin resistance), and MedDiet-Nuts (FoxO, PI3K-Akt, AMPK, p53 and HIF-1) interventions., Conclusion: Results show that 1-year MedDiet + Nuts and MedDiet + EVOO dietary interventions modulate exosomal RNA content, with the former affecting a higher number of miRNAs. The modulation of exosomal RNAs could help explain how the adherence to a Mediterranean diet may lead to beneficial effects and deserves further investigation., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

The field of epitranscriptomics is rapidly developing. Several modifications (e.g. methylations) have been identified for different RNA types. Current evidence shows that chemical RNA modifications can influence the whole molecule's secondary structure, translatability, functionality, stability, and degradation, and some are dynamically and reversibly modulated. miRNAs, in particular, are not only post-transcriptional modulators of gene expression but are themselves submitted to regulatory mechanisms. Understanding how these modifications are regulated and the resulting pathological consequences when dysregulation occurs is essential for the development of new therapeutic targets. In humans and other mammals, dietary components have been shown to affect miRNA expression and may also induce chemical modifications in miRNAs. The identification of chemical modifications in miRNAs (endogenous and exogenous) that can impact host gene expression opens up an alternative way to select new specific therapeutic targets.Hence, the aim of this review is to briefly address how RNA epitranscriptomic modifications can affect miRNA biogenesis and to summarize the existing evidence showing the connection between the (de)regulation of these processes and disease settings. In addition, we hypothesize on the potential effect certain chemical modifications could have on the potential cross-kingdom journey of dietary plant miRNAs.

Diet is a well-known risk factor of cardiovascular diseases (CVDs). Some microRNAs (miRNAs) have been described to regulate molecular pathways related to CVDs. Diet can modulate miRNAs and their target genes. Choline, betaine, and l-carnitine, nutrients found in animal products, are metabolized into trimethylamine n -oxide (TMAO), which has been associated with CVD risk. The aim of this study was to investigate TMAO regulation of CVD-related miRNAs and their target genes in cellular models of liver and macrophages. We treated HEPG-2, THP-1, mouse liver organoids, and primary human macrophages with 6 µM TMAO at different timepoints (4, 8, and 24 h for HEPG-2 and mouse liver organoids, 12 and 24 h for THP-1, and 12 h for primary human macrophages) and analyzed the expression of a selected panel of CVD-related miRNAs and their target genes and proteins by real-time PCR and Western blot, respectively. HEPG-2 cells were transfected with anti-miR-30c and syn-miR-30c. TMAO increased the expression of miR-21-5p and miR-30c-5p. PER2 , a target gene of both, decreased its expression with TMAO in HEPG-2 and mice liver organoids but increased its mRNA expression with syn-miR-30c. We concluded that TMAO modulates the expression of miRNAs related to CVDs, and that such modulation affects their target genes.

Broccoli ( Brassica oleracea var. italica) and its bioactive compounds are associated with beneficial health effects, which might be enabled, at least in part, through miRNA regulation, despite recent controversial studies suggesting that exogenous dietary miRNAs may reach host circulation and target cells to regulate gene expression. Here, a computational analysis was performed to explore the processes and pathways associated with genes targeted either by (1) host-expressed miRNAs (endogenous) modulated by the bioactive compounds in broccoli or (2) miRNAs derived from broccoli (exogenous). In addition, the stability of exogenous miRNAs from broccoli was assessed after broccoli was subjected to the usual processing methods and in vitro digestion-simulating gastrointestinal (GI) conditions. Overall, bioinformatic results show that the anticarcinogenic and cancer-preventive properties attributed to cruciferous vegetables might be mediated, at least in part, through miRNA-related mechanisms. Moreover, results show that broccoli-derived miRNAs can survive common food-processing conditions and GI digestion.

MicroRNAs (miRNAs) are small non-coding RNAs with a known role as mediators of gene expression in crucial biological processes, which converts them into high potential contenders in the ongoing search for effective therapeutic strategies. However, extracellular RNAs are unstable and rapidly degraded, reducing the possibility of successfully exerting a biological function in distant target cells. Strategies aimed at enhancing the therapeutic potential of miRNAs include the development of efficient, tissue-specific and nonimmunogenic delivery methods. Since miRNAs were discovered to be naturally transported within exosomes, a type of extracellular vesicle that confers protection against RNase degradation and increases miRNA stability have been proposed as ideal delivery vehicles for miRNA-based therapy. Although research in this field has grown rapidly in the last few years, a standard, reproducible and cost-effective protocol for exosome isolation and extracellular RNA delivery is lacking. We aimed to evaluate the use of milk-derived extracellular vesicles as vehicles for extracellular RNA drug delivery. With this purpose, exosomes were isolated from raw bovine milk, combining ultracentrifugation and size exclusion chromatography (SEC) methodology. Isolated exosomes were then loaded with exogenous hsa-miR148a-3p, a highly expressed miRNA in milk exosomes. The suitability of exosomes as delivery vehicles for extracellular RNAs was tested by evaluating the absorption of miR-148a-3p in hepatic (HepG2) and intestinal (Caco-2) cell lines. The potential exertion of a biological effect by miR-148a-3p was assessed by gene expression analysis, using microarrays. Results support that bovine milk is a cost-effective source of exosomes which can be used as nanocarriers of functional miRNAs with a potential use in RNA-based therapy. In addition, we show here that a combination of ultracentrifugation and SEC technics improve exosome enrichment, purity, and integrity for subsequent use., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The role of miRNAs in intestinal lipid metabolism is poorly described. The small intestine is constantly exposed to high amounts of dietary lipids, and it is under conditions of stress that the functions of miRNAs become especially pronounced. Approaches consisting in either a chronic exposure to cholesterol and triglyceride rich diets (for several days or weeks) or an acute lipid challenge were employed in the search for intestinal miRNAs with a potential role in lipid metabolism regulation. According to our results, changes in miRNA expression in response to fat ingestion are dependent on factors such as time upon exposure, gender and small intestine section. Classic and recent intestinal in vitro models (i.e. differentiated Caco-2 cells and murine organoids) partially mirror miRNA modulation in response to lipid challenges in vivo. Moreover, intestinal miRNAs might play a role in triglyceride absorption and produce changes in lipid accumulation in intestinal tissues as seen in a generated intestinal Dicer1-deletion murine model. Overall, despite some variability between the different experimental cohorts and in vitro models, results show that some miRNAs analysed here are modulated in response to dietary lipids, hence likely to participate in the regulation of lipid metabolism, and call for further research.

Consumption of highly processed foods, such as those high in trans fats and free sugars, coupled with sedentarism and chronic stress increases the risk of obesity and cardiometabolic disorders, while adherence to a Mediterranean diet is inversely associated with the prevalence of such diseases. Olive oil is the main source of fat in the Mediterranean diet. Data accumulated thus far show consumption of extra virgin, (poly)phenol-rich olive oil to be associated with specific health benefits. Of note, recommendations for consumption based on health claims refer to the phenolic content of extra virgin olive oil as beneficial. However, even though foods rich in monounsaturated fatty acids, such as olive oil, are healthier than foods rich in saturated and trans fats, their inordinate use can lead to adverse effects on health. The aim of this review was to summarize the data on olive oil consumption worldwide and to critically examine the literature on the potential adverse effects of olive oil and its main components, particularly any effects on lipid metabolism. As demonstrated by substantial evidence, extra virgin olive oil is healthful and should be preferentially used within the context of a balanced diet, but excessive consumption may lead to adverse consequences., (© The Author(s) 2020. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In support of the J curve that describes the association between wine consumption and all-cause mortality, researchers and the lay press often advocate the health benefits of (poly)phenol consumption via red wine intake and cite the vast amount of in vitro literature that would corroborate the hypothesis. Other researchers dismiss such evidence and call for total abstention. In this review, we take a skeptical, Pythagorean stance and we critically try to move the debate forward by pointing the readers to the many pitfalls of red wine (poly)phenol research, which we arbitrarily treat as if they were pharmacological agents. We conclude that, after 30 years of dedicated research and despite the considerable expenditure, we still lack solid, "pharmacological", human evidence to confirm wine (poly)phenols' biological actions. Future research will eventually clarify their activities and will back the current recommendations of responsibly drinking moderate amounts of wine with meals.

MicroRNAs (miRNAs) crucial roles in translation repression and post-transcriptional adjustments contribute to regulate intestinal lipid metabolism. Even though their actions in different metabolic tissues have been elucidated, their intestinal activity is yet unclear. We aimed to investigate intestinal miRNA-regulated lipid metabolism-related genes, by creating an intestinal-specific Dicer1 knockout (Int-Dicer1 KO) mouse model, with a depletion of microRNAs in enterocytes. The levels of 83 cholesterol and lipoprotein metabolism-related genes were assessed in the intestinal mucosa of Int-Dicer1 KO and Wild Type C57BL/6 (WT) littermates mice at baseline and 2 h after an oral lipid challenge. Among the 18 genes selected for further validation, Hmgcs2 , Acat1 and Olr1 were found to be strong candidates to be modulated by miRNAs in enterocytes and intestinal organoids. Moreover, we report that intestinal miRNAs contribute to the regulation of intestinal epithelial differentiation. Twenty-nine common miRNAs found in the intestines were analyzed for their potential to target any of the three candidate genes found and validated by miRNA-transfection assays in Caco-2 cells. MiR-31-5p, miR-99b-5p, miR-200a-5p, miR-200b-5p and miR-425-5p are major regulators of these lipid metabolism-related genes. Our data provide new evidence on the potential of intestinal miRNAs as therapeutic targets in lipid metabolism-associated pathologies., (Copyright © 2020 Ruiz-Roso, Gil-Zamorano, López de las Hazas, Tomé-Carneiro, Crespo, Latasa, Briand, Sánchez-López, Ortiz, Visioli, Martínez and Dávalos.)

Cognitive decline is one of the hallmarks of aging and can vary from mild cognitive impairment to dementia to Alzheimer's disease. In addition to some lifestyle interventions, there is room for the use of nutraceuticals/functional foods as pharma-nutritional tools to lessen the burden of cognitive decline before it worsens. We previously reported the promising molecular actions of milk fat globule membranes and krill oil concentrates in a rat model of aging. In this study, we concentrated on the activities on cognition, using an array of validated tests. We also performed lipidomic analyses of plasma, erythrocytes, and different brain areas. We report lower emotional memory (contextual fear conditioning) in aged rats supplemented with concentrates of polar lipids from buttermilk or krill oil at doses that approximate human consumption. No other behavioral parameter was significantly influenced by the supplements, calling for further research to confirm or not the purported salubrious activities of polar lipids, namely those rich in ω3 long-chain polyunsaturated fatty acids, on cognitive decline., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Hydroxytyrosol (HT) is involved in healthful activities and is beneficial to lipid metabolism. Many investigations focused on finding tissue-specific targets of HT through the use of different omics approaches such as transcriptomics and proteomics. However, it is not clear which (if any) of the potential molecular targets of HT reported in different studies are concurrently affected in various tissues. Following the bioinformatic analyses of publicly available data from a selection of in vivo studies involving HT-supplementation, we selected differentially expressed lipid metabolism-related genes and proteins common to more than one study, for validation in rodent liver samples from the entire selection. Four miRNAs (miR-802-5p, miR-423-3p, miR-30a-5p, and miR-146b-5p) responded to HT supplementation. Of note, miR-802-5p was commonly regulated in the liver and intestine. Our premise was that, in an organ crucial for lipid metabolism such as the liver, consistent modulation should be found for a specific target of HT even if different doses and duration of HT supplementation were used in vivo. Even though our results show inconsistency regarding differentially expressed lipid metabolism-related genes and proteins across studies, we found Fgf21 and Rora as potential novel targets of HT. Omics approaches should be fine-tuned to better exploit the available databases.

Postprandial lipemia has many physiopathological effects, some of which increase the risk of cardiovascular disease. MicroRNAs (miRNAs) can be found in almost all biological fluids, but their postprandial kinetics are poorly described. We aimed to profile circulating miRNAs in response to a fat challenge. In total, 641 circulating miRNAs were assessed by real-time PCR in plasmas from mice two hours after lipid gavage. Mice with intestine-specific loss of Dicer were screened to identify potential miRNAs released by the intestine. A total of 68 miRNAs were selected for further validation. Ten circulating miRNAs were finally validated as responsive to postprandial lipemia, including miR-206-3p, miR-543-3p, miR-466c-5p, miR-27b-5p, miR-409-3p, miR-340-3p, miR-1941-3p, miR-10a-3p, miR-125a-3p, and miR-468-3p. Analysis of their possible tissues of origin/target showed an enrichment of selected miRNAs in liver, intestine, brain, or skeletal muscle. miR-206, miR-27b-5p, and miR-409-3p were validated in healthy humans. Analysis of their predicted target genes revealed their potential involvement in insulin/insulin like growth factor (insulin/IGF), angiogenesis, cholecystokinin B receptor signaling pathway (CCKR), inflammation or Wnt pathways for mice, and in platelet derived growth factor (PDGF) and CCKR signaling pathways for humans. Therefore, the current study shows that certain miRNAs are released in the circulation in response to fatty meals, proposing them as potential novel therapeutic targets of lipid metabolism.

Impaired glucose metabolism and mitochondrial decay greatly increase with age, when cognitive decline becomes rampant. No pharmacological or dietary intervention has proven effective, but proper diet and lifestyle do postpone the onset of neurodegeneration and some nutrients are being investigated. We studied insulin signaling, mitochondrial activity and biogenesis, and synaptic signaling in the hippocampus and cortex following dietary supplementation with bioactive phospholipid concentrates of krill oil (KOC), buttermilk fat globule membranes (BMFC), and a combination of both in aged rats. After 3 months of supplementation, although all groups of animals showed clear signs of peripheral insulin resistance, the combination of KOC and BMFC was able to improve peripheral insulin sensitivity. We also explored brain energy balance. Interestingly, the hippocampus of supplemented rats-mainly when supplemented with BMFC or the combination of KOC and BMFC-showed an increase in intracellular adenosine triphosphate (ATP) levels, whereas no difference was observed in the cerebral cortex. Moreover, we found a significant increase of brain-derived neurotrophic factor (BDNF) in the hippocampus of BMFC+KO animals. In summary, dietary supplementation with KOC and/or BMFC improves peripheral and central insulin resistance, suggesting that their administration could delay the onset of these phenomena. Moreover, n-3 fatty acids (FAs) ingested as phospholipids increase BDNF levels favoring an improvement in energy state within neurons and facilitating both mitochondrial and protein synthesis, which are necessary for synaptic plasticity. Thus, dietary supplementation with n-3 FAs could protect local protein synthesis and energy balance within dendrites, favoring neuronal health and delaying cognitive decline associated to age-related disrepair.

The Mediterranean diet has been long associated with improved cardiovascular prognosis, chemoprevention, and lower incidence of neurodegeneration. Of the multiple components of this diet, olive oil stands out because its use has historically been limited to the Mediterranean basin. The health benefits of olive oil and some of its components are being rapidly decoded. In this paper we review the most recent pharma-nutritional investigations on olive oil biophenols and their health effects, chiefly focusing on recent findings that elucidate their molecular mechanisms of action.

The possibility that diet-derived miRNAs survive the gastrointestinal tract and exert biological effects in target cells is triggering considerable research in the potential abilities of alimentary preventive and therapeutic approaches. Many validation attempts have been carried out and investigators disagree on several issues. The barriers exogenous RNAs must surpass are harsh and adequate copies must reach target cells for biological actions to be carried out. This prospect opened a window for previously unlikely scenarios concerning exogenous non-coding RNAs, such as a potential role for breast milk microRNAs in infants' development and maturation. This review is focused on the thorny path breast milk miRNAs face towards confirmation as relevant role players in infants' development and maturation, taking into consideration the research carried out so far on the uptake, gastrointestinal barriers and potential biological effects of diet-derived miRNAs. We also discuss the future pharmacological and pharma-nutritional consequences of appropriate miRNAs research., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The increasing incidence of age-induced cognitive decline justifies the search for complementary ways of prevention or delay. We studied the effects of concentrates of phospholipids, sphingolipids, and/or 3-n fatty acids on the expression of genes or miRNAs related to synaptic activity and/or neurodegeneration, in the hippocampus of aged Wistar rats following a 3-month supplementation. The combination of two phospholipidic concentrates of krill oil (KOC) and buttermilk (BMFC) origin modulated the hippocampal expression of 119 miRNAs (11 were common to both BMFC and BMFC + KOC groups). miR-191a-5p and miR-29a-3p changed significantly only in the BMFC group, whereas miR-195-3p and miR-148a-5p did so only in the combined-supplemented group. Thirty-eight, 58, and 72 differentially expressed genes (DEG) were found in the groups supplemented with KOC, BMFC and BMFC + KOC, respectively. Interaction analysis unveiled networks of selected miRNAs with their potential target genes. DEG found in the KOC and BMFC groups were mainly involved in neuroactive processes, whereas they were associated with lysosomes and mRNA surveillance pathways in the BMFC + KOC group. We also report a significant reduction in hippocampal ceramide levels with BMFC + KOC. Our results encourage additional in-depth investigations regarding the potential beneficial effects of these compounds.

Background and Aims: Hydroxytyrosol (HT) is the primary phenolic compound of olives, virgin olive oil, and their byproducts. Proteomic analysis of metabolically active tissues helps elucidating novel mechanisms of action and potential targets in cardiometabolic disease. Thus, we aimed at determining the impact of long-term HT supplementation on the proteome of adipose and liver tissue, in mice., Methods: C57BL/6J mice received either a control diet or a diet supplemented with nutritionally relevant doses of HT for eight weeks., Results: HT supplementation differentially affects the adipose and liver tissues proteome, as evaluated by super-SILAC. Some oxidative stress-related proteins were modulated in both tissues, such as the multifunctional protein peroxiredoxin 1, which was consistently repressed by HT supplementation. In some cases tissue-dependent modulation was observed, as in the case of FASN., Conclusions: This study provides interesting information on the connection between changes seen at tissue proteome level and the metabolic effects of HT. The use of this pertinent proteomics quantification approach may prove quite useful for uncovering novel potential pharmaco-nutritional targets of HT supplementation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Recent epidemiological evidence demonstrated that diabetes is a risk factor for AD onset and development. Indeed, meta-analyses of longitudinal epidemiologic studies show that diabetes increases AD risk by 50-100%, being insulin resistance (IR) the main binding link between diabetes and AD. Astrocytes are the foremost cerebral macroglial cells and are responsible for converting glucose into lactate and transfer it to neurons that use it as fuel, but Aβ(1-42) impairs insulin signaling and glycogen storage. Recent prospective studies showed that the Mediterranean diet is associated with lower incidence of AD. We hypothesized that hydroxytyrosol (HT, the preeminent polyphenol of olives and olive oil) could exert beneficial effects on IR associated with AD and investigated it mechanisms of action in an astrocytic model of AD. The astrocytic cell line C6 was exposed to Aβ(25-35) and co-incubated with HT for different periods. After treatment with Aβ(25-35), astrocytes' viability was significantly decreased as compared with controls; however, both pre- and post-treatment with HT prevented this effect. Mechanistically, we found that the preventive role of HT on Aβ(25-35)- induced cytotoxicity in astrocytes is moderated by an increased HT-induced activation of Akt, which is mediated by the insulin signaling pathway. In addition, we report that HT prevented the pronounced activation of mTOR, thereby restoring proper insulin signaling. In conclusion, we demonstrate that HT protects Aβ(25-35)-treated astrocytes by improving insulin sensitivity and restoring proper insulin-signaling. These data provide some mechanistic insight on the observed inverse association between olive oil consumption and prevalence of cognitive impairment. © 2017 BioFactors, 43(4):540-548, 2017., (© 2017 International Union of Biochemistry and Molecular Biology.)

Background: In addition to prescription drugs, nutraceuticals/functional foods/medical foods are being increasingly added as adjunct treatment of cardiovascular disease (CVD), even though most of them have been exclusively studied in vitro., Hypothesis/purpose: We review the available evidence (focusing on when the amount of polyphenols' intake was measured) coming from randomized controlled trials (RCTs) of (poly)phenol-based supplements., Conclusion: We conclude that (poly)phenol-based nutraceuticals and functional foods might be indeed used as adjunct therapy of CVD, but additional long-term RCTs with adequate numerosity and with clinically relevant end points are needed to provide unequivocal evidence of their clinical usefulness., (Copyright © 2015. Published by Elsevier GmbH.)

Dietary microRNAs (miRNAs) modulation could be important for health and wellbeing. Part of the healthful activities of polyphenols might be due to a modulation of miRNAs' expression. Among the most biologically active polyphenols, hydroxytyrosol (HT) has never been studied for its actions on miRNAs. We investigated whether HT could modulate the expression of miRNAs in vivo. We performed an unbiased intestinal miRNA screening in mice supplemented (for 8 weeks) with nutritionally relevant amounts of HT. HT modulated the expression of several miRNAs. Analysis of other tissues revealed consistent HT-induced modulation of only few miRNAs. Also, HT administration increased triglycerides levels. Acute treatment with HT and in vitro experiments provided mechanistic insights. The HT-induced expression of one miRNA was confirmed in healthy volunteers supplemented with HT in a randomized, double-blind and placebo-controlled trial. HT consumption affects specific miRNAs' expression in rodents and humans. Our findings suggest that the modulation of miRNAs' action through HT consumption might partially explain its healthful activities and might be pharmanutritionally exploited in current therapies targeting endogenous miRNAs. However, the effects of HT on triglycerides warrant further investigations., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Scope: Ellagitannins, ellagic acid, and the colonic metabolites urolithins (Uros) exhibit anticancer effects against colon cells, but a comprehensive molecular analysis has not been done. Herein, we used a panel of cell lines to first time evaluate the antiproliferative properties and accompanying molecular responses of two ellagitannin metabolites mixtures mimicking the situation in vivo and of each individual metabolite., Methods and Results: We examined cell growth, cell cycle, apoptosis, and the expression of related genes and microRNAs (miRs) in a panel of nonmalignant and malignant colon cell lines. Regardless of the composition, the mixed metabolites similarly inhibited proliferation, induced cycle arrest, and apoptosis. All the metabolites contributed to these effects, but Uro-A, isourolithin A, Uro-C, and Uro-D were more potent than Uro-B and ellagic acid. Despite molecular differences between the cell lines, we discerned relevant changes in key cancer markers and corroborated the induction of CDKN1A (cyclin-dependent kinase inhibitor 1A gene (p21, Cip1); encoding p21) as a common step underlying the anticancer properties of Uros. Interestingly, cell-unique downregulation of miR-224 or upregulation of miR-215 was found associated with CDKN1A induction., Conclusion: Physiologically relevant mixtures of Uros exert anticancer effects against colon cancer cells via a common CDKN1A upregulatory mechanism. Other associated molecular responses are however heterogeneous and mostly cell-specific., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chemotherapy increases the overall survival in colorectal cancer (CRC) patients. 5-Fluorouracil (5-FU) remains as a drug of first choice in CRC therapy over the last four decades. However, only 10-15% of patients with advanced CRC respond positively to 5-FU monotherapy. Therefore, new strategies to enhance the 5-FU effectiveness, overcome the tumor cell resistance and decrease the unspecific toxicity are critically needed. Urolithin A (Uro-A) is the main metabolite produced by the human gut microbiota from the dietary polyphenol ellagic acid. Uro-A targets the colonic mucosa of CRC patients, and preclinical studies have shown the anti-inflammatory and cancer chemopreventive activities of this metabolite. We evaluated here whether Uro-A, at concentrations achievable in the human colorectum, could sensitize colon cancer cells to 5-FU and 5'DFUR (a pro-drug intermediate of 5-FU). We found that both 5-FU and 5'DFUR arrested the cell cycle at the S phase by regulating cyclins A and B1 in the human colon cancer cells Caco-2, SW-480 and HT-29, and also triggered apoptosis through the activation of caspases 8 and 9. Co-treatments with Uro-A decreased IC50 values for both 5-FU and 5'DFUR and additionally arrested the cell cycle at the G2/M phase together with a slight increase in caspases 8 and 9 activation. Overall, we show that Uro-A potentiated the effects of both 5-FU and 5'DFUR on colon cancer cells. This suggests the need for lower 5-FU doses to achieve similar effects, which could reduce possible adverse effects. Further in vivo investigations are warranted to explore the possible role of Uro-A as a chemotherapy adjuvant.

The notion that (poly)phenols act as direct free radical scavengers is being challenged by mere chemical and biochemical considerations such as bioavailability and intracellular concentrations. An alternative hypothesis that is gaining considerable traction is that (poly)phenols are processed by the body as xenobiotics via the Keap1/Nrf2/ARE signaling axis, leading to the induction of Phase II enzymes. However, there are no solid human data to confirm this interesting supposition. In this study, we tested the activities of hydroxytyrosol (HT) on Phase II enzymes' expression in a double-blind, randomized, placebo-controlled study. We tested two HT doses, i.e. 5 and 25mg/d, vs. placebo following a Latin square design. We report that HT is well tolerated but does not significantly modify Phase II enzyme expression in peripheral blood mononuclear cells. Moreover, we were unable to record significant effects on a variety of surrogate markers of cardiovascular disease such as lipid profile and inflammation and oxidation markers. Available evidence indicates that the "hormesis hypothesis" that (poly)phenols activate Phase II enzymes requires solid human confirmation that might be provided by future trials. This study is registered at ClinicalTrials.gov (identifier: NCT02273622)., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Soy consumption has been suggested to afford protection from cardiovascular disease (CVD). Indeed, accumulated albeit controversial evidence suggests that daily consumption of ≥25 g of soy protein with its associated phytochemicals intact can improve lipid profiles in hypercholesterolemic humans. However, the belief that soy foods and supplements positively impact human health has become increasingly controversial among the general public because of the reported estrogenic activities of soy isoflavones. In this study, we investigated the nutrigenomic actions of soy isoflavones (in nutritionally-relevant amounts) with a specific focus on the adipose tissue, due to its pivotal role in cardiometabolism. Young C57BL/6 mice were maintained for eight weeks under two different diet regimes: (1) purified control diet; or (2) purified control diet supplemented with 0.45 g% soybean dry purified extract (a genistein/daidzein mix). Soy isoflavones increased plasma total cholesterol concentrations and decreased triglyceride ones. Circulating leptin levels was also increased by soy consumption. Differentially expressed genes in adipose tissue were classified according to their role(s) in cellular or metabolic pathways. Our data show that soy isoflavones, administered in nutritionally-relevant amounts, have diverse nutrigenomic effects on adipose tissue. Taking into account the moderate average exposure to such molecules, their impact on cardiovascular health needs to be further investigated to resolve the issue of whether soy consumption does indeed increase or decrease cardiovascular risk.

Primary prevention of cardiovascular disease (CVD) aims to avoid a first event in subjects that are at risk but have not yet been diagnosed with heart disease. Secondary prevention of CVD aims to avoid new events in patients with established heart disease. Both approaches involve clinical intervention and implementation of healthy lifestyles. The grape and wine polyphenol resveratrol (3,5,4'-trihydroxy-trans-stilbene) has shown cardioprotective benefits in humans. Most of these approaches deal with rather high doses and short follow-ups, and do not address the issue of long-term resveratrol consumption safety, especially in medicated individuals. Here, we review the trials conducted with resveratrol in patients at risk for or with established CVD, focusing on the two longest human clinical trials reported so far (1-year follow-up). We also discuss the expectations for resveratrol from a dietary and clinical perspective in relation to CVD. However, statistically significant changes in CVD-risk markers do not necessarily equal clinical significance in the daily care of patients., (© 2013 New York Academy of Sciences.)

Numerous studies have shown that resveratrol (RES) exerts anti-inflammatory effects but human trials evidencing these effects in vivo are limited. Furthermore, the molecular mechanisms triggered in humans following the oral intake of RES are not yet understood. Therefore, the purpose of this study was to investigate the molecular changes in peripheral blood mononuclear cells (PBMCs) associated to the one-year daily intake of a RES enriched (8 mg) grape extract (GE-RES) in hypertensive male patients with type 2 diabetes mellitus (T2DM). We used microarrays and RT-PCR to analyze expression changes in genes and microRNAs (miRs) involved in the inflammatory response modulated by the consumption of GE-RES in comparison to a placebo and GE lacking RES. We also examined the changes in several serobiochemical variables, inflammatory and fibrinolytic markers. Our results showed that supplementation with GE or GE-RES did not affect body weight, blood pressure, glucose, HbA1c or lipids, beyond the values regulated by gold standard medication in these patients. We did not find either any significant change on serum inflammatory markers except for a significant reduction of ALP and IL-6 levels. The expression of the pro-inflammatory cytokines CCL3, IL-1β and TNF-α was significantly reduced and that of the transcriptional repressor LRRFIP-1 increased in PBMCs from patients taking the GE-RES extract. Also, a group of miRs involved in the regulation of the inflammatory response: miR-21, miR-181b, miR-663, miR-30c2, miR-155 and miR-34a were found to be highly correlated and altered in the group consuming the GE-RES for 12 months. Our results provide preliminary evidence that long-term supplementation with a grape extract containing RES downregulates the expression of key pro-inflammatory cytokines with the involvement of inflammation-related miRs in circulating immune cells of T2DM hypertensive medicated patients and support a beneficial immunomodulatory effect in these patients., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: The grape and wine polyphenol resveratrol exerts cardiovascular benefits but evidence from randomized human clinical trials is very limited. We investigated dose-depending effects of a resveratrol-containing grape supplement on stable patients with coronary artery disease (CAD) treated according to currently accepted guidelines for secondary prevention of cardiovascular disease., Methods: In a triple-blind, randomized, placebo-controlled, one-year follow-up, 3-arm pilot clinical trial, 75 stable-CAD patients received 350 mg/day of placebo, resveratrol-containing grape extract (grape phenolics plus 8 mg resveratrol) or conventional grape extract lacking resveratrol during 6 months, and a double dose for the following 6 months. Changes in circulating inflammatory and fibrinolytic biomarkers were analyzed. Moreover, the transcriptional profiling of inflammatory genes in peripheral blood mononuclear cells (PBMCs) was explored using microarrays and functional gene expression analysis., Results: After 1 year, in contrast to the placebo and conventional grape extract groups, the resveratrol-containing grape extract group showed an increase of the anti-inflammatory serum adiponectin (9.6 %, p = 0.01) and a decrease of the thrombogenic plasminogen activator inhibitor type 1 (PAI-1) (-18.6 %, p = 0.05). In addition, 6 key inflammation-related transcription factors were predicted to be significantly activated or inhibited, with 27 extracellular-space acting genes involved in inflammation, cell migration and T-cell interaction signals presenting downregulation (p < 0.05) in PBMCs. No adverse effects were detected in relation to the study products., Conclusions: Chronic daily consumption of a resveratrol-containing grape nutraceutical could exert cardiovascular benefits in stable-CAD patients treated according to current evidence-based standards, by increasing serum adiponectin, preventing PAI-1 increase and inhibiting atherothrombotic signals in PBMCs.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a non-flavonoid polyphenol that may be present in a limited number of foodstuffs such as grapes and red wine. Resveratrol has been reported to exert a plethora of health benefits through many different mechanisms of action. This versatility and presence in the human diet have drawn the worldwide attention of many research groups over the past twenty years, which has resulted in a huge output of in vitro and animal (preclinical) studies. In line with this expectation, many resveratrol- based nutraceuticals are consumed all over the world with questionable clinical/scientific support. In fact, the confirmation of these benefits in humans through randomized clinical trials is still very limited. The vast majority of preclinical studies have been performed using assay conditions with a questionable extrapolation to humans, i.e. too high concentrations with potential safety concerns (adverse effects and drug interactions), short-term exposures, in vitro tests carried out with non-physiological metabolites and/or concentrations, etc. Unfortunately, all these hypothesis-generating studies have contributed to increased the number of 'potential' benefits and mechanisms of resveratrol but confirmation in humans is very limited. Therefore, there are many issues that should be addressed to avoid an apparent endless loop in resveratrol research. The so-called 'Resveratrol Paradox', i.e., low bioavailability but high bioactivity, is a conundrum not yet solved in which the final responsible actor (if any) for the exerted effects has not yet been unequivocally identified. It is becoming evident that resveratrol exerts cardioprotective benefits through the improvement of inflammatory markers, atherogenic profile, glucose metabolism and endothelial function. However, safety concerns remain unsolved regarding chronic consumption of high RES doses, specially in medicated people. This review will focus on the currently available evidence regarding resveratrol's effects on humans obtained from randomized clinical trials. In addition, we will provide a critical outlook for further research on this molecule that is evolving from a minor dietary compound to a possible multi-target therapeutic drug.

Scope: The cardioprotective role of resveratrol as part of the human diet is not yet clear. Our aim was to investigate the effect of a grape supplement containing 8 mg resveratrol in oxidized LDL (LDLox), apolipoprotein-B (ApoB), and serum lipids on statin-treated patients in primary cardiovascular disease prevention (PCP)., Methods and Results: A triple-blind, randomized, placebo-controlled trial was conducted. Seventy-five patients (three parallel arms) consumed one capsule (350 mg) daily for 6 months containing resveratrol-enriched grape extract (GE-RES, Stilvid®), grape extract (GE, similar polyphenolic content but no resveratrol), or placebo (maltodextrin). After 6 months, no changes were observed in the placebo group and only LDL cholesterol (LDLc) decreased by 2.9% (p = 0.013) in the GE group. In contrast, LDLc (-4.5%, p = 0.04), ApoB (-9.8%, p = 0.014), LDLox (-20%, p = 0.001), and LDLox/ApoB (-12.5%, p = 0.000) decreased in the Stilvid® group, whereas the ratio non-HDLc (total atherogenic cholesterol load)/ApoB increased (8.5%, p = 0.046). No changes were observed in hepatic, thyroid, and renal function. No adverse effects were observed in any of the patients., Conclusion: This GE-RES reduced atherogenic markers and might exert additional cardioprotection beyond the gold-standard medication in patients from PCP. The presence of resveratrol in the GE was necessary to achieve these effects., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

There is no pharmaceutical or definitive surgical cure for inflammatory bowel diseases (IBDs). The naturally occurring polyphenol resveratrol exerts anti-inflammatory properties. However, its rapid metabolism diminishes its effectiveness in the colon. The design of prodrugs to targeting active molecules to the colon provides an opportunity for therapy of IBDs. Herein we explore the efficacy of different resveratrol prodrugs and pro-prodrugs to ameliorate colon inflammation in the murine dextran sulfate sodium (DSS) model. Mice fed with a very low dose (equivalent to 10 mg for a 70 kg-person) of either resveratrol-3-O-(6'-O-butanoyl)-β-D-glucopyranoside (6) or resveratrol-3-O-(6'-O-octanoyl)-β-D-glucopyranoside (7) did not develop colitis symptoms and improved 6-fold the disease activity index (DAI) compared to resveratrol. Our results indicate that these pro-prodrugs exerted a dual effect: (1) they prevented the rapid metabolism of resveratrol and delivered higher quantities of resveratrol to the colon and (2) they reduced mucosal barrier imbalance and prevented diarrhea, which consequently facilitated the action of the delivered resveratrol in the colon mucosa.