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Stress disorders have dramatically increased in recent decades becoming the most prevalent psychiatric disorder in the United States and Europe. However, the diagnosis of stress disorders is currently based on symptom checklist and psychological questionnaires, thus making the identification of candidate biomarkers necessary to gain better insights into this pathology and its related metabolic alterations. Regarding the identification of potential biomarkers, omic profiling and metabolic footprint arise as promising approaches to recognize early biochemical changes in such disease and provide opportunities for the development of integrative candidate biomarkers. Here, we studied plasma and urine metabolites together with metagenomics in a 3 days Chronic Unpredictable Mild Stress (3d CUMS) animal approach that aims to focus on the early stress period of a well-established depression model. The multi-omics integration showed a profile composed by a signature of eight plasma metabolites, six urine metabolites and five microbes. Specifically, threonic acid, malic acid, alpha-ketoglutarate, succinic acid and cholesterol were proposed as key metabolites that could serve as key potential biomarkers in plasma metabolome of early stages of stress. Such findings targeted the threonic acid metabolism and the tricarboxylic acid (TCA) cycle as important pathways in early stress. Additionally, an increase in opportunistic microbes as virus of the Herpesvirales was observed in the microbiota as an effect of the primary stress stages. Our results provide an experimental biochemical characterization of the early stage of CUMS accompanied by a subsequent omic profiling and a metabolic footprinting that provide potential candidate biomarkers.

Obesity is one of the most incident and concerning disease worldwide. Definite strategies to prevent obesity and related complications remain elusive. Among the risk factors of the onset of obesity, gut microbiota might play an important role in the pathogenesis of the disease, and it has received extensive attention because it affects the host metabolism. In this study, we aimed to define a metabolic profile of the segregated obesity-associated gut dysbiosis risk factor. The study of the metabolome, in an obesity-associated gut dysbiosis model, provides a relevant way for the discrimination on the different biomarkers in the obesity onset. Thus, we developed a model of this obesity risk factors through the transference of gut microbiota from obese to non-obese male Wistar rats and performed a subsequent metabolic analysis in the receptor rats. Our results showed alterations in the lipid metabolism in plasma and in the phenylalanine metabolism in urine. In consequence, we have identified metabolic changes characterized by: (1) an increase in DG:34:2 in plasma, a decrease in hippurate, (2) an increase in 3-HPPA, and (3) an increase in o-coumaric acid. Hereby, we propose these metabolites as a metabolic profile associated to a segregated dysbiosis state related to obesity disease.

The increasing prevalence of obesity has become a worldwide health concern. Currently, obesity is tackled through caloric restriction and physical activity, and in some cases, with pharmacotherapy. Even so, these strategies have shown to not be ideal in some cases, and other alternatives, such as the use of natural bioactive compounds, are being studied. Mitochondria are importantly affected in obesity, particularly in the skeletal muscle, the liver, and the white and brown adipose tissues. Thus, researchers are focusing on the study of natural bioactive compounds, such as polyphenols, which could target specifically the mitochondrial function, to improve obesity. This chapter provides a summary of the evidences for the use of bioactive compounds including polyphenols such as resveratrol or naringenin, and other compounds such as omega-3 polyunsaturated fatty acids and retinoids, to correct or prevent obesity through their effects on mitochondrial modulation.

The metabolic syndrome is a multifactorial disease developed due to accumulation and chronification of several risk factors associated with disrupted metabolism. The early detection of the biomarkers by NMR spectroscopy could be helpful to prevent multifactorial diseases. The exposure of each risk factor can be detected by traditional molecular markers but the current biomarkers have not been enough precise to detect the primary stages of disease. Thus, there is a need to obtain novel molecular markers of pre-disease stages. A promising source of new molecular markers are metabolomics standing out the research of biomarkers in NMR approaches. An increasing number of nutritionists integrate metabolomics into their study design, making nutrimetabolomics one of the most promising avenues for improving personalized nutrition. This review highlight the major five risk factors associated with metabolic syndrome and related diseases including carbohydrate dysfunction, dyslipidemia, oxidative stress, inflammation, and gut microbiota dysbiosis. Together, it is proposed a profile of metabolites of each risk factor obtained from NMR approaches to target them using personalized nutrition, which will improve the quality of life for these patients.

Deregulation of miR-33 and miR-122, as major regulators of lipid metabolism in liver, has been related to obesity and metabolic syndrome. Proanthocyanidins repress these microRNAs in healthy animals. Hence, we hypothesized that long-term consumption of dietary proanthocyanidins can normalize the expression of miR-33a and miR-122. Therefore, the objective of this work was to determine whether the long-term consumption of proanthocyanidins could effectively normalize the expression of miR-33a and miR-122 in rats made obese by a high-fat diet and to determine the effective dose. Rats were maintained on the high-fat diet with or without supplementation with a grape seed proanthocyanidin extract at low, medium, or high dose in relation to human consumption. Results show that 3 weeks of supplementation with grape seed proanthocyanidin extract normalized the overexpression of miR-33a and miR-122 in obese rats' liver for all doses studied, with no dose-dependent outcome, and also reduced the levels of plasma and liver lipids in a dose-dependent manner. In conclusion, a low sustained dose of proanthocyanidins, lower than the estimated mean intake for a European population, is enough to normalize miR-33a and miR-122 levels in the livers of obese rats. Therefore, a proanthocyanidin-rich diet during obesity can improve some of the metabolic syndrome symptoms at least at the molecular level.

Circadian rhythm plays an important role in maintaining homeostasis, and its disruption increases the risk of developing metabolic syndrome. Circadian rhythm is maintained by a central clock in the hypothalamus that is entrained by light, but circadian clocks are also present in peripheral tissues. These peripheral clocks are trained by other cues, such as diet. The aim of this study was to determine whether proanthocyanidins, the most abundant polyphenols in the human diet, modulate the expression of clock and clock-controlled genes in the liver, gut and mesenteric white adipose tissue (mWAT) in healthy and obese rats. Grape seed proanthocyanidin extracts (GSPEs) were administered for 21 days at 5, 25 or 50 mg GSPE/kg body weight in healthy rats and 25 mg GSPE/kg body weight in rats with diet-induced obesity. In healthy animals, GSPE administration led to the overexpression of core clock genes in a positive dose-dependent manner. Moreover, the acetylated BMAL1 protein ratio increased with the same pattern in the liver and mWAT. With regards to clock-controlled genes, Per2 was also overexpressed, whereas Rev-erbα and RORα were repressed in a negative dose-dependent manner. Diet-induced obesity always resulted in the overexpression of some core clock and clock-related genes, although the particular gene affected was tissue specific. GSPE administration counteracted disturbances in the clock genes in the liver and gut but was less effective in normalizing the clock gene disruption in WAT. In conclusion, proanthocyanidins have the capacity to modulate peripheral molecular clocks in both healthy and obese states.

Postprandial lipemia influences the development of atherosclerosis, which itself constitutes a risk factor for the development of cardiovascular diseases. The introduction of bioactive compounds may prevent these deleterious effects. Proanthocyanidins are potent antioxidants that have hypolipidemic properties, while omega-3 polyunsaturated fatty acids (ω3 PUFAs) stimulate fatty acid oxidation and gene expression programs, controlling mitochondrial functions. In this study, we investigated the effects of acute treatment of ω3 PUFAs and proanthocyanidins on the metabolic flexibility and lipid handling profiles in the skeletal muscle and adipose tissue of rats that were raised on diets, high in saturated fatty acids. For this, oil rich in docosahexaenoic (DHA-OR), grape seed proanthocyanidins extract (GSPE), or both substances (GSPE + DHA-OR) were administered with an overload of lard oil to healthy Wistar rats. Our results indicate that the addition of DHA-OR to lard oil increases insulin sensitivity and redirects fatty acids toward skeletal muscle, thereby activating fatty acid oxidation. We also observed an improvement in adipose mitochondrial functionality and uncoupling. In contrast, GSPE lowers lipidemia, prevents muscle reactive oxygen species (ROS) production and damage, furthermore, activates mitochondrial biogenesis and lipogenesis in adipose tissue. The addition of GSPE+DHA-OR to lard resulted in nearly all the effects of DHA-OR and GSPE administered individually, but the combined administration resulted in a more attenuated profile. © 2013 BioFactors, 40(1):146–156, 2014

Metabolism follows circadian rhythms, which are driven by peripheral clocks. Clock genes in the liver are entrained by daytime meals and food components. Proanthocyanidins (PAs), the most abundant flavonoids in the human diet, modulate lipid and glucose metabolism. The aim of this study was to determine whether PAs could adjust the clock system in the liver. Male Wistar rats were orally gavaged with 250 mg grape seed proanthocyanidin extract (GSPE)/kg body weight at zeitgeber time (ZT) 0 (light turned on), at ZT12 (light turned off), or before a 6 hour jet-lag and sacrificed at different times. The 24 hour rhythm of clock-core and clock-controlled gene expression indicated that nicotinamide phosphoribosyltransferase (Nampt) was the most sensitive gene to GSPE. However, Nampt was repressed or overexpressed after GSPE administration at ZT0 or ZT12, respectively. NAD levels, which are controlled by Nampt and also exhibit circadian rhythm, decreased or increased according to Nampt expression. Moreover, the ratio of acetylated Bmal1, that directly drives Nampt expression, only increased when GSPE was administered at ZT12. Therefore, GSPE modulated the clock system in the liver, suggesting that PAs can regulate lipid and glucose metabolism by adjusting the circadian rhythm in the liver.

Dietary proanthocyanidins modulate melatonin levels in plasma and the expression pattern of clock genes in the hypothalamus of rats Scope: Circadian rhythms allow organisms to anticipate and adapt to environmental changes, and food components can adjust internal rhythms. Proanthocyanidins improve cardiovascular risk factors that exhibit circadian oscillations. Therefore, the aim of the current study was to determine whether proanthocyanidins can modulate body rhythms. Methods and results: Male Wistar rats were orally gavaged with 250 mg grape seed proanthocyanidin extract (GSPE)/kg body weight at zeitgeber time (ZT) 0 (light on). Phenotypic biorhythm was evaluated by measuring the concentration of plasma melatonin and metabolites, using MNR-metabolomics, at several ZT. Remarkably, GSPE treatment maintained nocturnal melatonin levels at ZT3 and altered the oscillations of some metabolites in plasma. Quantification of expression of clock-core (Clock, Bmal1, Per2, Rorα, Rev-erbα) and clock-controlled (Nampt) genes in the hypothalamus by RT-PCR showed that this phenotypic alteration was concomitant with the modulation of the expression pattern of Bmal1 and Nampt. However, GSPE did not modulate the nocturnal expression of clock genes when administered at ZT12 (light off). Conclusion: PAs could have chronobiological properties, although their activity depends largely on the time of administration. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In the lasts years it has become evident that polyphenols modify cell functionality through epigenetic mechanisms, such as modulating microRNA (miRNA) levels. miRNAs are small non-coding RNAs of about 22 nucleotides in length, that modulate gene expression at the post-transcriptional level. miRNAs are involved in almost all biological processes, affect most metabolic pathways and recent evidence suggests their dysregulation in a number of metabolic disorders and diseases. In this sense, miRNAs are emerging as potential biomarkers of numerous pathologies and therefore as new therapeutic targets. Polyphenolic modulation of miRNAs is very attractive as a strategy to target numerous cell processes and potentially reduce the risk of chronic diseases.

Obesity has become a worldwide epidemic. The cafeteria diet (CD) induces obesity and oxidative-stress-associated insulin resistance. Polyunsaturated fatty acids and polyphenols are dietary compounds that are intensively studied as products that can reduce the health complications related to obesity. We evaluate the effects of 21 days of supplementation with grape seed proanthocyanidins extract (GSPE), docosahexaenoic-rich oil (DHA-OR) or both compounds (GSPE+DHA-OR) on skeletal muscle metabolism in diet-obese rats. The supplementation with different treatments did not reduce body weight, although all groups used more fat as fuel, particularly when both products were coadministered; muscle β-oxidation was activated, the mitochondrial functionality and oxidative capacity were higher, and fatty acid uptake gene expressions were up-regulated. In addition to these outcomes shared by all treatments, GSPE reduced insulin resistance and improved muscle status. Both treatments increased 5’-AMP-activated protein kinase (AMPK) phosphorylation, which was consistent with higher plasma adiponectin levels. Moreover, AMPK activation by DHA-OR was also correlated with an up-regulation of peroxisome proliferator-activated receptor alpha (Pparα). GSPE+DHA-OR, in addition to activating AMPK and enhancing fatty acid oxidation, increased the muscle gene expression of uncoupling protein 2 (Ucp2). In conclusion, GSPE+DHA-OR induced modifications that improved muscle status and could counterbalance the deleterious effects of obesity, and such modifications are mediated, at least in part, through the AMPK signaling pathway.

Skeletal muscle is a key organ of mammalian energy metabolism, and its mitochondria are multifunction organelles that are targets of dietary bioactive compounds. The goal of this work was to examine the regulation of mitochondrial dynamics, functionality and cell energy parameters using docosahexaenoic acid (DHA), epigallocatechin gallate (EGCG) and a combination of both in L6 myocytes. Compounds (at 25μM) were incubated for 4h. Cells cultured with DHA displayed less oxygen consumption with higher ADP/ATP ratio levels concomitant with downregulation of Cox and Ant1 gene expression. The disruption of energetic homeostasis by DHA, increases intracellular reactive oxygen species (ROS) levels and decreases mitochondrial membrane potential. The defence mechanism to counteract the excess of ROS production was by the upregulation of Ucp2, Ucp3 and MnSod gene expression. Moreover myocytes cultured with DHA had a higher mitochondrial mass with a higher proportion of large and elongated mitochondria, whereas the fission genes Drp1 and Fiss1 and the fusion gene Mfn2 were downregulated. In myocytes co-incubated with DHA and EGCG, ROS levels and the adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio were similar to untreated myocytes and the decrease of oxygen consumption, higher mitochondrial mass and the overexpression of Ucp2 and Ucp3 genes were similar to the DHA-treated cells with also a higher amount of mitochondrial deoxyribonucleic acid (DNA), and reduced Drp1 and Fiss1 gene expression levels. In conclusion the addition of EGCG to DHA returned the cells to the control conditions in terms of mitochondrial morphology, energy and redox status, which were unbalanced in the DHA-treated myocytes.

Modulation of miR-33 and miR-122 has been proposed to be a promising strategy to treat dyslipidemia and insulin resistance associated with obesity and metabolic syndrome. Interestingly, specific polyphenols reduce the levels of these mi(cro)RNAs. The aim of this study was to elucidate the effect of polyphenols of different chemical structure on miR-33a and miR-122 expression and to determine whether direct binding of the polyphenol to the mature microRNAs (miRNAs) is a plausible mechanism of modulation. The effect of two grape proanthocyanidin extracts, their fractions and pure polyphenol compounds on miRNA expression was evaluated using hepatic cell lines. Results demonstrated that the effect on miRNA expression depended on the polyphenol chemical structure. Moreover, miR-33a was repressed independently of its host-gene SREBP2. Therefore, the ability of resveratrol and epigallocatechin gallate to bind miR-33a and miR-122 was measured using (1)H NMR spectroscopy. Both compounds bound miR-33a and miR-122 and differently. Interestingly, the nature of the binding of these compounds to the miRNAs was consistent with their effects on cell miRNA levels. Therefore, the specific and direct binding of polyphenols to miRNAs emerges as a new posttranscriptional mechanism by which polyphenols could modulate metabolism.

10.1016/j.jnutbio.2013.09.014 Elevated postprandial triglycerides are associated with an increased risk of cardiovascular disease. Acute proanthocyanidin supplementation improves postprandial lipemia. Therefore, in this study, we evaluated whether a chronic treatment (3weeks) of grape seed proanthocyanidins (GSPE) improves tolerance to lipid overload and represses liver miRNA-33a and miRNA-122 and their target genes as a mechanism to soften the elevated postprandial triglycerides in healthy rats. Additionally, the minimal GSPE chronic dose required to alter miRNA levels was determined by means of a dose-response experiment using 5, 15, 25, or 50mg of GSPE/kg body weight. GSPE repressed miR-33a and miR-122 liver expression and reduced postprandial lipemia in a dose-dependent manner. Significant effects were only observed at high levels of proanthocyanidin consumption, but moderate doses of proanthocyanidins were still able to modulate miRNA expression. Therefore, it can be suggested that a population with a normal intake of proanthocyanidin-rich foods can benefit from the modulation of miRNA expression. At the molecular level, this action can confer homeostatic robustness and will thus exert subtle changes in lipid metabolism, thereby reducing the risk associated with postprandial hyperlipemia.

miR-33 and miR-122 are major regulators of lipid metabolism in the liver, and their deregulation has been linked to the development of metabolic diseases such as obesity and metabolic syndrome. However, the biological importance of these miRNAs has been defined using genetic models. The aim of this study was to evaluate whether the levels of miR-122 and miR-33a in rat liver correlate with lipemia in nutritional models. For this purpose, we analyzed the levels of miRNA-33a and miR-122 in the livers of dyslipidemic cafeteria diet-fed rats and of cafeteria diet-fed rats supplemented with proanthocyanidins and/or ω-3 PUFAs because these two dietary components are well-known to counteract dyslipidemia. The results showed that the dyslipidemia induced in rats that were fed a cafeteria diet resulted in the upregulation of miR-33a and miR-122 in the liver, whereas the presence of proanthocyanidins and/or ω-3 PUFAs counteracted the increase of these two miRNAs. However, srebp2, the host gene of miR-33a, was significantly repressed by ω-3 PUFAs but not by proanthocyanidins. Liver mRNA levels of the miR-122 and miR-33a target genes, fas and pparβ/δ, cpt1a and abca1, respectively, were consistent with the expression of these two miRNAs under each condition. Moreover, the miR-33a and abca1 levels were also analyzed in PBMCs. Interestingly, the miR-33a levels evaluated in PBMCs under each condition were similar to the liver levels but enhanced. This demonstrates that miR-33a is expressed in PBMCs and that these cells can be used as a non-invasive way to reflect the expression of this miRNA in the liver. These findings cast new light on the regulation of miR-33a and miR-122 in a dyslipidemic model of obese rats and the way these miRNAs are modulated by dietary components in the liver and in PBMCs.

MicroRNAs (miRNAs) are small noncoding RNAs, approximately 18-25 nucleotides in length, that modulate gene expression at the posttranscriptional level. Thousands of miRNAs have been described, and it is thought that they regulate some aspects of more than 60% of all human cell transcripts. Several polyphenols have been shown to modulate miRNAs related to metabolic homeostasis and chronic diseases. Polyphenolic modulation of miRNAs is very attractive as a strategy to target numerous cell processes and potentially reduce the risk of chronic disease. Evidence is building that polyphenols can target specific miRNAs, such as miR-122, but more studies are necessary to discover and validate additional miRNA targets.

Scope One major health problem in westernized countries is dysregulated fatty acid and cholesterol metabolism that causes pathologies such as metabolic syndrome. Previous studies from our group have shown that proanthocyanidins, which are the most abundant polyphenols in the human diet, regulate lipid metabolism and are potent hypolipidemic agents. The noncoding RNAs, miR-33 and miR-122, regulate genes that are involved in lipid metabolism. Methods and results Here, we show that grape seed proanthocyanidins rapidly and transiently repressed the expression of miR-33 and miR-122 in rat hepatocytes in vivo and in vitro. Furthermore, the miR-33 target gene ATP-binding cassette A1 and the miR-122 target gene fatty acid synthase were also modulated by proanthocyanidins. Specifically, ATP-binding cassette A1 mRNA and protein levels were increased, and fatty acid synthase mRNA and protein levels were reduced after the miRNA levels were altered. Conclusion These results suggest that proanthocyanidin treatment increased hepatic cholesterol efflux to produce new HDL particles by repressing miR-33, and it reduced lipogenesis by repressing miR-122. These results highlight a new mechanism by which grape seed proanthocyanidins produce hypolipidemia through their effects on miRNA modulators of lipid metabolism.

DOI: 10.1016/j.tifs.2016.11.019 URL: http://www.sciencedirect.com/science/article/pii/S0924224416302357 Filiació URV: SI Inclòs a la memòria: SI Background Non-alcoholic fatty liver disease (NAFLD) can be regarded as the hepatic manifestation of metabolic syndrome (MS), which has a high prevalence in Western-lifestyle countries. NAFLD can progress to more severe pathologies such as cirrhosis and fibrosis, thus worsening individuals’ life quality and increasing health costs. In the last decades, many efforts have been made in order to understand the molecular mechanisms of NAFLD and how to cure it. It has become clear that alimentary regimen is of primary importance for preventing/treating MS and NAFLD. Scope and approach Beneficial effects of the Mediterranean diet (MedDiet) on MS and NAFLD are now recognized and can be ascribed to low calories intake but also to abundance of anti-oxidant and anti-inflammatory compounds which are present in fruit and vegetables as well as in herbs and spices widely used to flavor traditional Mediterranean dishes. The aim of this review is to summarize briefly NAFLD molecular pathways and therapies, while focusing on the beneficial effects and mechanisms of action of the most used Mediterranean aromatic plants and spices (MAPS). Key findings and conclusions All MAPS reviewed here have been documented to exert hepatoprotective actions and almost the totality has a direct or indirect lipid-lowering effect on the liver, accompanied by amelioration of other MS parameters. We suggest that culinary MAPS use can contribute to the health benefits of MedDiet without the danger of adverse effects that can occur when the active compounds are used in pharmacological doses.