Fiber from elicited butternut pumpkin (Cucurbita moschata D. cv. Ariel) modulates the human intestinal microbiota dysbiosis.

Elicited pumpkin was evaluated as a potential daily consumption product able to modulate the gut microbiota. An in vitro dynamic colonic fermentation performance with microbiota from obese volunteers was used. Prebiotic effects were observed after the pumpkin treatment. Bifidobacterium abundance was maintained during the treatment period whereas Lactobacillus increased in the transversal and descending colon. Conversely, Enterobacteriaceae and Clostridium groups were more stable, although scarce decreasing trends were observed for same species. Increments of Lactobacillus acidophilus and Limosilactobacillus fermentum (old Lactobacillus fermentum) were observed in the whole colonic tract after the treatment period. However, modulatory effects were mainly observed in the transversal and descending colon. Diverse bacteria species were increased, such as Akkermansia muciniphila, Bacteroides dorei, Cloacibacillus porcorum, Clostridium lactatifermentans, Ruminococcus albus, Ruminococcus lactaris, Coprococcus catus, Alistipes shahii or Bacteroides vulgatus. The prebiotic effect of the elicited pumpkin was provided by the fiber of the pumpkin, suggesting a release of pectin molecules in the transversal and distal colonic tract through low cellulosic fiber degradation, explaining the increases in the total propionic and butyric acid in these colonic sections. Also, a possible modulatory role of carotenoids from the sample was suggested since carotenes were found in the descending colon. Hence, the results of this research highlighted pumpkin as a natural product able to modulate the microbiota towards a healthier profile.

Isoliquiritigenin in combination with visceral adipose tissue and related markers as a predictive tool for nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver disease in the world. New non-invasive diagnostic tools are needed to promptly treat this disease and avoid its complications. This study aimed to find key metabolites and related variables that could be used to predict and diagnose NAFLD. Ninety-eight subjects with NAFLD and 45 controls from the Fatty Liver in Obesity (FLiO) Study (NCT03183193) were analyzed. NAFLD was diagnosed and graded by ultrasound and classified into two groups: 0 (controls) and ≥ 1 (NAFLD). Hepatic status was additionally assessed through magnetic resonance imaging (MRI), elastography, and determination of transaminases. Anthropometry, body composition (DXA), biochemical parameters, and lifestyle factors were evaluated as well. Non-targeted metabolomics of serum was performed with high-performance liquid chromatography coupled to time-of-flight mass spectrometry (HPLC-TOF-MS). Isoliquiritigenin (ISO) had the strongest association with NAFLD out of the determinant metabolites. Individuals with higher concentrations of ISO had healthier metabolic and hepatic status and were less likely to have NAFLD (OR 0.13). Receiver operating characteristic (ROC) curves demonstrated the predictive power of ISO in panel combination with other NAFLD and IR-related variables, such as visceral adipose tissue (VAT) (AUROC 0.972), adiponectin (AUROC 0.917), plasmatic glucose (AUROC 0.817), and CK18-M30 (AUROC 0.810). Individuals with lower levels of ISO have from 71 to 82% more risk of presenting NAFLD compared to individuals with higher levels. Metabolites such as ISO, in combination with visceral adipose tissue, IR, and related markers, constitute a potential non-invasive tool to predict and diagnose NAFLD.

Impact of acute consumption of beverages containing plant-based or alternative sweetener blends on postprandial appetite, food intake, metabolism, and gastro-intestinal symptoms: Results of the SWEET beverages trial.

Project SWEET examined the barriers and facilitators to the use of non-nutritive sweeteners and sweetness enhancers (hereafter "S&SE") alongside potential risks/benefits for health and sustainability. The Beverages trial was a double-blind multi-centre, randomised crossover trial within SWEET evaluating the acute impact of three S&SE blends (plant-based and alternatives) vs. a sucrose control on glycaemic response, food intake, appetite sensations and safety after a carbohydrate-rich breakfast meal. The blends were: mogroside V and stevia RebM; stevia RebA and thaumatin; and sucralose and acesulfame-potassium (ace-K). At each 4 h visit, 60 healthy volunteers (53% male; all with overweight/obesity) consumed a 330 mL beverage with either an S&SE blend (0 kJ) or 8% sucrose (26 g, 442 kJ), shortly followed by a standardised breakfast (∼2600 or 1800 kJ with 77 or 51 g carbohydrates, depending on sex). All blends reduced the 2-h incremental area-under-the-curve (iAUC) for blood insulin (p < 0.001 in mixed-effects models), while the stevia RebA and sucralose blends reduced the glucose iAUC (p < 0.05) compared with sucrose. Post-prandial levels of triglycerides plus hepatic transaminases did not differ across conditions (p > 0.05 for all). Compared with sucrose, there was a 3% increase in LDL-cholesterol after stevia RebA-thaumatin (p < 0.001 in adjusted models); and a 2% decrease in HDL-cholesterol after sucralose-ace-K (p < 0.01). There was an impact of blend on fullness and desire to eat ratings (both p < 0.05) and sucralose-acesulfame K induced higher prospective intake vs sucrose (p < 0.001 in adjusted models), but changes were of a small magnitude and did not translate into energy intake differences over the next 24 h. Gastro-intestinal symptoms for all beverages were mostly mild. In general, responses to a carbohydrate-rich meal following consumption of S&SE blends with stevia or sucralose were similar to sucrose.

An In Vitro Protocol to Study the Modulatory Effects of a Food or Biocompound on Human Gut Microbiome and Metabolome.

The gut microbiota plays a key role in gastrointestinal immune and metabolic functions and is influenced by dietary composition. An in vitro protocol simulating the physiological conditions of the digestive system helps to study the effects of foods/biocompounds on gut microbiome and metabolome. The Dynamic-Colonic Gastrointestinal Digester consists of five interconnected compartments, double jacket vessels that simulate the physiological conditions of the stomach, the small intestine and the three colonic sections, which are the ascending colon, transverse colon and descending colon. Human faeces are required to reproduce the conditions and culture medium of the human colon, allowing the growth of the intestinal microbiota. After a stabilization period of 12 days, a food/biocompound can be introduced to study its modulatory effects during the next 14 days (treatment period). At the end of the stabilization and treatment period, samples taken from the colon compartments are analysed. The 16S rRNA gene analysis reveals the microbiota composition. The untargeted metabolomics analysis gives more than 10,000 features (metabolites/compounds). The present protocol allows in vitro testing of the modulatory effects of foods or biocompounds on gut microbiota composition and metabolic activity.

Diet- and sex-related changes of gut microbiota composition and functional profiles after 4 months of weight loss intervention.

PURPOSE: Obesity has been related to intestinal dysbiosis and the modification of gut microbiota composition by dietary strategies becomes a promising strategy to help manage obesity. The aim of the current study was to evaluate the effect of two weight-loss diets on the composition and functional profile of gut microbiota. METHODS: 55 men and 124 women with BMI > 25 kg/m2 were randomly assigned to moderately high-protein (MHP) or low-fat (LF) diet. Differences in fecal bacteria abundance (based on 16 s rRNA sequencing) between before and after 4 months of calorie restriction was analyzed using EdgeR tool in MicrobiomeAnalyst platform. Bacterial functional profile was predicted using Tax4Fun and metagenomeSeq analysis. Significant KEGG Orthology (KO) terms were selected for the metabolomic study using chromatography. RESULTS: After the intervention, MHP-men showed a significant decrease in Negativicutes, Selenomonadales, Dielma and Dielma fastidiosa. LF-men showed a significant increase in Bacilli, Lactobacillales, Christensenellaceae, Peptococcaceae, and Streptococcaceae, Peptococcus, Streptococcus and Christensenella, Duncaniella dubosii_CP039396_93.49%, Roseburia sp_AB744234_98.96% and Alistipes inops_KJ572413_99.57%. MHP-women increased Pasteurellales, Phascolarctobacterium succinatutens, Ruthenibacterium lactatiformans_LR215981_99.55% and decreased in Phascolarctobacterium succinatutens_NR112902_99.56%. Finally, LF-women presented a significant decrease in Bacteroides clarus and Erysipelothrix inopinata_CP060715_84.4%. Surprisingly, no matching bacterial changes were found between these four groups. A total of 42 KO, 10 metabolic pathways and 107 related metabolites related were found implicated in these bacterial changes. Seven metabolites were confirmed in plasma. CONCLUSION: Weight-loss-related-changes in gut microbiome composition and the functional profile occur in a sex- and diet-related manner, showing that women and men could differentially benefit from the consumption of MHP and LF diets. TRIAL REGISTRATION: NCT02737267, 10th March 2016 retrospectively registered.

Phenolic Compounds Reduce the Fat Content in Caenorhabditis elegans by Affecting Lipogenesis, Lipolysis, and Different Stress Responses.

Supplementation with bioactive compounds capable of regulating energy homeostasis is a promising strategy to manage obesity. Here, we have screened the ability of different phenolic compounds (myricetin, kaempferol, naringin, hesperidin, apigenin, luteolin, resveratrol, curcumin, and epicatechin) and phenolic acids (p-coumaric, ellagic, ferulic, gallic, and vanillic acids) regulating C. elegans fat accumulation. Resveratrol exhibited the strongest lipid-reducing activity, which was accompanied by the improvement of lifespan, oxidative stress, and aging, without affecting worm development. Whole-genome expression microarrays demonstrated that resveratrol affected fat mobilization, fatty acid metabolism, and unfolded protein response of the endoplasmic reticulum (UPRER), mimicking the response to calorie restriction. Apigenin induced the oxidative stress response and lipid mobilization, while vanillic acid affected the unfolded-protein response in ER. In summary, our data demonstrates that phenolic compounds exert a lipid-reducing activity in C. elegans through different biological processes and signaling pathways, including those related with lipid mobilization and fatty acid metabolism, oxidative stress, aging, and UPR-ER response. These findings open the door to the possibility of combining them in order to achieve complementary activity against obesity-related disorders.

A combination of borage seed oil and quercetin reduces fat accumulation and improves insulin sensitivity in obese rats.

The metabolic properties of omega-6 fatty acid consumption are being increasingly accepted. We had previously observed that supplementation with a borage seed oil (BSO), as a source of linoleic (18:2n-6; LA) and gamma-linolenic (18:3n-6; GLA) acids, reduces body weight and visceral adiposity and improves insulin sensitivity in a diet-induced obesity model of Wistar rats. Here, it was investigated whether the anti-obesogenic properties of BSO could be maintained in a pre-obese model of rats, and if these effects are enhanced by a combination with low doses of quercetin, together with its potential role in the regulation of the adipocyte biology. The combination of BSO and quercetin during 8 weeks was able to ameliorate glucose intolerance and insulin resistance, and to improve liver steatosis. Although no effects were observed on body weight, animals supplemented with this combination exhibited a lower proportion of visceral adiposity. In addition, in vitro differentiation of epididymal adipose-precursor cells of the BSO-treated animals exhibited a down-regulation of Fasn, Glut4, Pparg and Srebp1 genes, in comparison with the control group. Finally, in vitro evaluation of the components of BSO demonstrated that the anti-adipogenic activity of quercetin was significantly potentiated by the combination with both LA and GLA through the down-regulation of different adipogenesis-key genes in 3T3-L1 cells. All these data suggest that omega-6 fatty acids LA and GLA, and their natural sources such as BSO, could be combined with quercetin to potentiate their effects in the prevention of the excess of adiposity and the insulin resistance.

Low doses of cocoa extract supplementation ameliorate diet-induced obesity and insulin resistance in rats.

Cocoa polyphenols exhibit high antioxidant activity and have been proposed as a potential adjuvant for the treatment of metabolic disturbances. Here, we demonstrate that supplementation with low doses (14 and 140 mg per kg per rat) of a complete cocoa extract induces metabolic benefits in a diet-induced obesity (DIO) model of Wistar rats. After 10 weeks, cocoa extract-supplemented animals exhibited significantly lower body weight gain and food efficiency, with no differences in energy intake. Cocoa significantly reduced visceral (epididymal and retroperitoneal) and subcutaneous fat accumulation accompanied by a significant reduction in the adipocyte size, which was mediated by downregulation of the adipocyte-specific genes Cebpa, Fasn and Adipoq. Additionally, cocoa extract supplementation reduced the triacylglycerol/high density lipoprotein (TAG/HDL) ratio, decreased hepatic triglyceride accumulation, improved insulin sensitivity by reducing HOMA-IR, and significantly ameliorated glucose tolerance after an intraperitoneal glucose tolerance test. Finally, no adverse effect was observed in an in vivo toxicity evaluation of our cocoa extract at doses up to 500 mg kg-1 day-1. Our data demonstrate that low doses of cocoa extract supplementation (14 and 140 mg kg-1 day-1) are safe and sufficient to counteract obesity and type-2 diabetes in rats and provide new insights into the potential application of cocoa supplements in the management of the metabolic syndrome.

Phenolic Compounds Inhibit 3T3-L1 Adipogenesis Depending on the Stage of Differentiation and Their Binding Affinity to PPARγ.

Phenolic compounds might modulate adiposity. Here, we report our observation that polyphenols and phenolic acids inhibit adipogenesis in 3T3-L1 with different intensity depending on the family and the stage of differentiation. While quercetin and resveratrol inhibited lipid accumulation along the whole process of differentiation, apigenin and myricetin were active during the early and latest stages, but not intermediate, contrary to hesperidin. The activity of phenolic acids was limited to the early stages of the differentiation process, except p-coumaric and ellagic acids. This anti-adipogenic effect was accompanied by down-regulation of Scd1 and Lpl. Molecular docking analysis revealed that the inhibitory activity of these phenolic compounds over the early stages of adipogenesis exhibits a significant correlation (r = 0.7034; p = 0.005) with their binding affinity to the ligand-binding domain of PPARγ. Results show that polyphenols and phenolic acids would interact with specific residues of the receptor, which could determine their potential anti-adipogenic activity during the early stages of the differentiation. Residues Phe264, His266, Ile281, Cys285 and Met348 are the most frequently involved in these interactions, which might suggest a crucial role for these amino acids modulating the activity of the receptor. These data contribute to elucidate the possible mechanisms of phenolic compounds in the control of adipogenesis.

Borago officinalis seed oil (BSO), a natural source of omega-6 fatty acids, attenuates fat accumulation by activating peroxisomal beta-oxidation both in C. elegans and in diet-induced obese rats.

Obesity is a medical condition with increasing prevalence, characterized by an accumulation of excess fat that could be improved using some bioactive compounds. However, many of these compounds with in vitro activity fail to respond in vivo, probably due to the sophistication of the physiological energy regulatory networks. In this context, C. elegans has emerged as a plausible model for the identification and characterization of the effect of such compounds on fat storage in a complete organism. However, the results obtained in such a simple model are not easily extrapolated to more complex organisms such as mammals, which hinders its application in the short term. Therefore, it is necessary to obtain new experimental data about the evolutionary conservation of the mechanisms of fat loss between worms and mammals. Previously, we found that some omega-6 fatty acids promote fat loss in C. elegans by up-regulation of peroxisomal fatty acid ß-oxidation in an omega-3 independent manner. In this work, we prove that the omega-6 fatty acids' effects on worms are also seen when they are supplemented with a natural omega-6 source (borage seed oil, BSO). Additionally, we explore the anti-obesity effects of two doses of BSO in a diet-induced obesity rat model, validating the up-regulation of peroxisomal fatty acid ß-oxidation. The supplementation with BSO significantly reduces body weight gain and energy efficiency and prevents white adipose tissue accumulation without affecting food intake. Moreover, BSO also increases serum HDL-cholesterol levels, improves insulin resistance and promotes the down-regulation of Cebpa, an adipogenesis-related gene. Therefore, we conclude that the effects of omega-6 fatty acids are highly conserved between worms and obesity-induced mammals, so these compounds could be considered to treat or prevent obesity-related disorders.

Untargeted metabolomic on urine samples after α-lipoic acid and/or eicosapentaenoic acid supplementation in healthy overweight/obese women.

BACKGROUND: Eicosapentaenoic acid (EPA) and α-lipoic acid (α-LA) have been investigated for their beneficial effects on obesity and cardiovascular risk factors. In the current research, the goal was to evaluate metabolomic changes following the dietary supplementation of these two lipids, alone or combined in healthy overweight/obese sedentary women following an energy-restricted diet. For this purpose, an untargeted metabolomics approach was conducted on urine samples using liquid chromatography coupled with time of flight mass spectrometry (HPLC-TOF-MS). METHODS: This is a short-term double blind placebo-controlled study with a parallel nutritional design that lasted 10 weeks. Participants were assigned to one of the 4 experimental groups [Control, EPA (1.3 g/d), α-LA (0.3 g/d) and EPA+α-LA (1.3 g/d + 0.3 g/d)]. All intervention groups followed an energy-restricted diet of 30% less than total energy expenditure. Clinically relevant biochemical measurements were analyzed. Urine samples (24 h) were collected at baseline and after 10 weeks. Untargeted metabolomic analysis on urine samples was carried out, and principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were performed for the pattern recognition and characteristic metabolites identification. RESULTS: Urine samples were scattered in the PCA scores plots in response to the supplementation with α-LA. Totally, 28 putative discriminant metabolites in positive ionization, and 6 in negative ionization were identified among groups clearly differentiated according to the α-LA administration. Remarkably is the presence of an ascorbate intermediate metabolite (one of the isomers of trihydroxy-dioxohexanoate, or dihydroxy-oxohexanedionate) in the groups supplemented with α-LA. This fact might be associated with antioxidant properties of both α-LA and ascorbic acid. Correlations between phenotypical parameters and putative metabolites of provided additional information on whether there is a direct or inverse relationship between them. Especially interesting are the negative correlation between ascorbate intermediate metabolite and asymmetric dimethylarginine (ADMA) and the positive one between superoxide dismutase (SOD) and α-LA supplementation. CONCLUSIONS: This metabolomic approach supports that the beneficial effects of α-LA administration on body weight reduction may be partly explained by the antioxidant properties of this organosulfur carboxylic acid mediated by isomers of trihydroxy-dioxohexanoate, or dihydroxy-oxohexanedionate. TRIAL REGISTRATION: Clinicaltrials.gov NCT01138774 .

Dihomo-gamma-linolenic acid induces fat loss in C. elegans in an omega-3-independent manner by promoting peroxisomal fatty acid ß-oxidation.

Bioactive compounds, including some fatty acids (FAs), can induce beneficial effects on body fat-content and metabolism. In this work, we have used C. elegans as a model to examine the effects of several FAs on body fat accumulation. Both omega-3 and omega-6 fatty acids induced a reduction of fat content in C. elegans, with linoleic, gamma-linolenic and dihomo-gamma-linolenic acids being the most effective ones. These three FAs are sequential metabolites especially in omega-6 PUFA synthesis pathway and the effects seem to be primarily due to dihomo-gamma-linolenic acid, and independent of its transformation into omega-3 or arachidonic acid. Gene expression analyses suggest that peroxisomal beta oxidation is the main mechanism involved in the observed effect. These results point out the importance of further analysis of the activity of these omega-6 FAs, due to their potential application in obesity and related diseases.

Freeze-dried strawberry and blueberry attenuates diet-induced obesity and insulin resistance in rats by inhibiting adipogenesis and lipogenesis.

Obesity and type 2-diabetes are becoming a worldwide health problem, reiterating the importance of alternative therapies to tackle their progression. Here, we hypothesized that supplementation of diet with 6% w/w of a freeze-dried strawberry-blueberry (5 : 1) powder (FDSB) could exert beneficial metabolic effects on Wistar rats. FDSB-supplemented animals experienced significantly reduced body weight gain, food efficiency and visceral adiposity accumulation in two independent experiments. FDSB supplementation also contributed to lower area under the curve after an intraperitoneal GTT and reduced serum insulin levels and an insulin resistance index (IR-HOMA) in HFS diet-fed animals, together with reduced plasma MCP-1 inflammation marker concentrations. Gene expression analysis in retroperitoneal adipocytes from experiment 1 and 3T3-L1 cells showed that FDSB inhibited adipogenesis and lipogenesis through down-regulation of Pparg, Cebpa, Lep, Fasn, Scd-1 and Lpl gene expression. Untargeted metabolomics identified the cis isomer of resveratrol-3-glucoside-sulphate as a metabolite differentially increased in FDSB-treated serum samples, which corresponds to a strawberry metabolite that could be considered a serum biomarker of FDSB-intake. Our results suggest that FDSB powder might be useful for treatment/prevention of obesity-related diseases.

The urinary metabolomic profile following the intake of meals supplemented with a cocoa extract in middle-aged obese subjects.

Metabolomics is used to assess the compliance and bioavailability of food components, as well as to evaluate the metabolic changes associated with food consumption. This study aimed to analyze the effect of consuming ready-to-eat meals containing a cocoa extract, within an energy restricted diet on urinary metabolomic changes. Fifty middle-aged volunteers [30.6 (2.3) kg m(-2)] participated in a 4-week randomised, parallel and double-blind study. Half consumed meals supplemented with 1.4 g of cocoa extract (645 mg polyphenols) while the remaining subjects received meals without cocoa supplementation. Ready-to-eat meals were included within a 15% energy restricted diet. Urine samples (24 h) were collected at baseline and after 4 weeks and were analyzed by high-performance-liquid chromatography-time-of-flight-mass-spectrometry (HPLC-TOF-MS) in negative and positive ionization modes followed by multivariate analysis. The relationship between urinary metabolites was evaluated by the Spearman correlation test. Interestingly, the principal component analysis discriminated among the baseline group, control group at the endpoint and cocoa group at the endpoint (p < 0.01), although in the positive ionization mode the baseline and control groups were not well distinguished. Metabolites were related to theobromine metabolism (3-methylxanthine and 3-methyluric acid), food processing (L-beta-aspartyl-L-phenylalanine), flavonoids (2,5,7,3',4'-pentahydroxyflavanone-5-O-glucoside and 7,4'-dimethoxy-6-C-methylflavanone), catecholamine (3-methoxy-4-hydroxyphenylglycol-sulphate) and endogenous metabolism (uridine monophosphate). These metabolites were present in higher (p < 0.001) amounts in the cocoa group. 3-Methylxanthine and l-beta-aspartyl-L-phenylalanine were confirmed with standards. Interestingly, 3-methoxy-4-hydroxyphenylglycol-sulphate was positively correlated with 3-methylxanthine (rho = 0.552; p < 0.001) and 7,4'-dimethoxy-6-C-methylflavanone (rho = 447; p = 0.002). In conclusion, the metabolomic approach supported the compliance of the volunteers with the intervention and suggested the bioavailability of cocoa compounds within the meals.

Metabolic faecal fingerprinting of trans-resveratrol and quercetin following a high-fat sucrose dietary model using liquid chromatography coupled to high-resolution mass spectrometry.

Faecal non-targeted metabolomics deciphers metabolic end-products resulting from the interactions among food, host genetics, and gut microbiota. Faeces from Wistar rats fed a high-fat sucrose (HFS) diet supplemented with trans-resveratrol and quercetin (separately or combined) were analysed by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). Metabolomics in faeces are categorised into four clusters based on the type of treatment. Tentative identification of significantly differing metabolites highlighted the presence of carbohydrate derivatives or conjugates (3-phenylpropyl glucosinolate and dTDP-D-mycaminose) in the quercetin group. The trans-resveratrol group was differentiated by compounds related to nucleotides (uridine monophosphate and 2,4-dioxotetrahydropyrimidine D-ribonucleotide). Marked associations between bacterial species (Clostridium genus) and the amount of some metabolites were identified. Moreover, trans-resveratrol and resveratrol-derived microbial metabolites (dihydroresveratrol and lunularin) were also identified. Accordingly, this study confirms the usefulness of omics-based techniques to discriminate individuals depending on the physiological effect of food constituents and represents an interesting tool to assess the impact of future personalized therapies.

Thermal protection of ß-carotene in re-assembled casein micelles during different processing technologies applied in food industry.

ß-Carotene is a carotenoid usually applied in the food industry as a precursor of vitamin A or as a colourant. ß-Carotene is a labile compound easily degraded by light, heat and oxygen. Casein micelles were used as nanostructures to encapsulate, stabilise and protect ß-carotene from degradation during processing in the food industry. Self-assembly method was applied to re-assemble nanomicelles containing ß-carotene. The protective effect of the nanostructures against degradation during the most common industrial treatments (sterilisation, pasteurisation, high hydrostatic pressure and baking) was proven. Casein micelles protected ß-carotene from degradation during heat stabilisation, high pressure processing and the processes most commonly used in the food industry including baking. This opens new possibilities for introducing thermolabile ingredients in bakery products.