Glucose-lowering effects of a synbiotic combination containing Pediococcus acidilactici in C. elegans and mice.

AIMS/HYPOTHESIS: Modulation of gut microbiota has emerged as a promising strategy to treat or prevent the development of different metabolic diseases, including type 2 diabetes and obesity. Previous data from our group suggest that the strain Pediococcus acidilactici CECT9879 (pA1c) could be an effective probiotic for regulating glucose metabolism. Hence, the objectives of this study were to verify the effectiveness of pA1c on glycaemic regulation in diet-induced obese mice and to evaluate whether the combination of pA1c with other normoglycaemic ingredients, such as chromium picolinate (PC) and oat ß-glucans (BGC), could increase the efficacy of this probiotic on the regulation of glucose and lipid metabolism. METHODS: Caenorhabditis elegans was used as a screening model to describe the potential synbiotic activities, together with the underlying mechanisms of action. In addition, 4-week-old male C57BL/6J mice were fed with a high-fat/high-sucrose diet (HFS) for 6 weeks to induce hyperglycaemia and obesity. Mice were then divided into eight groups (n=12 mice/group) according to dietary supplementation: control-diet group; HFS group; pA1c group (1010 colony-forming units/day); PC; BGC; pA1c+PC+BGC; pA1c+PC; and pA1c+BGC. Supplementations were maintained for 10 weeks. Fasting blood glucose was determined and an IPGTT was performed prior to euthanasia. Fat depots, liver and other organs were weighed, and serum biochemical variables were analysed. Gene expression analyses were conducted by real-time quantitative PCR. Sequencing of the V3-V4 region of the 16S rRNA gene from faecal samples of each group was performed, and differential abundance for family, genera and species was analysed by ALDEx2R package. RESULTS: Supplementation with the synbiotic (pA1c+PC+BGC) counteracted the effect of the high glucose by modulating the insulin-IGF-1 signalling pathway in C. elegans, through the reversal of the glucose nuclear localisation of daf-16. In diet-induced obese mice, all groups supplemented with the probiotic significantly ameliorated glucose tolerance after an IPGTT, demonstrating the glycaemia-regulating effect of pA1c. Further, mice supplemented with pA1c+PC+BGC exhibited lower fasting blood glucose, a reduced proportion of visceral adiposity and a higher proportion of muscle tissue, together with an improvement in the brown adipose tissue in comparison with the HFS group. Besides, the effect of the HFS diet on steatosis and liver damage was normalised by the synbiotic. Gene expression analyses demonstrated that the synbiotic activity was mediated not only by modulation of the insulin-IGF-1 signalling pathway, through the overexpression of GLUT-1 and GLUT-4 mediators, but also by a decreased expression of proinflammatory cytokines such as monocyte chemotactic protein-1. 16S metagenomics demonstrated that the synbiotic combinations allowed an increase in the concentration of P. acidilactici, together with improvements in the intestinal microbiota such as a reduction in Prevotella and an increase in Akkermansia muciniphila. CONCLUSIONS/INTERPRETATION: Our data suggest that the combination of pA1c with PC and BGC could be a potential synbiotic for blood glucose regulation and may help to fight insulin resistance, diabetes and obesity.

Effects of Glucosinolate-Enriched Red Radish (Raphanus sativus) on In Vitro Models of Intestinal Microbiota and Metabolic Syndrome-Related Functionalities.

The gut microbiota profile is determined by diet composition, and therefore this interaction is crucial for promoting specific bacterial growth and enhancing the health status. Red radish (Raphanus sativusL.) contains several secondary plant metabolites that can exert a protective effect on human health. Recent studies have shown that radish leaves have a higher content of major nutrients, minerals, and fiber than roots, and they have garnered attention as a healthy food or supplement. Therefore, the consumption of the whole plant should be considered, as its nutritional value may be of greater interest. The aim of this work is to evaluate the effects of glucosinolate (GSL)-enriched radish with elicitors on the intestinal microbiota and metabolic syndrome-related functionalities by using an in vitro dynamic gastrointestinal system and several cellular models developed to study the GSL impact on different health indicators such as blood pressure, cholesterol metabolism, insulin resistance, adipogenesis, and reactive oxygen species (ROS). The treatment with red radish had an influence on short-chain fatty acids (SCFA) production, especially on acetic and propionic acid and many butyrate-producing bacteria, suggesting that consumption of the entire red radish plant (leaves and roots) could modify the human gut microbiota profile toward a healthier one. The evaluation of the metabolic syndrome-related functionalities showed a significant decrease in the gene expression of endothelin, interleukin IL-6, and cholesterol transporter-associated biomarkers (ABCA1 and ABCG5), suggesting an improvement of three risk factors associated with metabolic syndrome. The results support the idea that the use of elicitors on red radish crops and its further consumption (the entire plant) may contribute to improving the general health status and gut microbiota profile.

Holistic Integration of Omics Tools for Precision Nutrition in Health and Disease.

The combination of multiple omics approaches has emerged as an innovative holistic scope to provide a more comprehensive view of the molecular and physiological events underlying human diseases (including obesity, dyslipidemias, fatty liver, insulin resistance, and inflammation), as well as for elucidating unique and specific metabolic phenotypes. These omics technologies include genomics (polymorphisms and other structural genetic variants), epigenomics (DNA methylation, histone modifications, long non-coding RNA, telomere length), metagenomics (gut microbiota composition, enterotypes), transcriptomics (RNA expression patterns), proteomics (protein quantities), and metabolomics (metabolite profiles), as well as interactions with dietary/nutritional factors. Although more evidence is still necessary, it is expected that the incorporation of integrative omics could be useful not only for risk prediction and early diagnosis but also for guiding tailored dietary treatments and prognosis schemes. Some challenges include ethical and regulatory issues, the lack of robust and reproducible results due to methodological aspects, the high cost of omics methodologies, and high-dimensional data analyses and interpretation. In this review, we provide examples of system biology studies using multi-omics methodologies to unravel novel insights into the mechanisms and pathways connecting the genotype to clinically relevant traits and therapy outcomes for precision nutrition applications in health and disease.

Grifola frondosa (Maitake) Extract Reduces Fat Accumulation and Improves Health Span in C. elegans through the DAF-16/FOXO and SKN-1/NRF2 Signalling Pathways.

In recent years, food ingredients rich in bioactive compounds have emerged as candidates to prevent excess adiposity and other metabolic complications characteristic of obesity, such as low-grade inflammation and oxidative status. Among them, fungi have gained popularity for their high polysaccharide content and other bioactive components with beneficial activities. Here, we use the C. elegans model to investigate the potential activities of a Grifola frondosa extract (GE), together with the underlying mechanisms of action. Our study revealed that GE represents an important source of polysaccharides and phenolic compounds with in vitro antioxidant activity. Treatment with our GE extract, which was found to be nongenotoxic through a SOS/umu test, significantly reduced the fat content of C. elegans, decreased the production of intracellular ROS and aging-lipofuscin pigment, and increased the lifespan of nematodes. Gene expression and mutant analyses demonstrated that the in vivo anti-obesity and antioxidant activities of GE were mediated through the daf-2/daf-16 and skn-1/nrf-2 signalling pathways, respectively. Taken together, our results suggest that our GE extract could be considered a potential functional ingredient for the prevention of obesity-related disturbances.

Differential response to a 6-month energy-restricted treatment depending on SH2B1 rs7359397 variant in NAFLD subjects: Fatty Liver in Obesity (FLiO) Study.

PURPOSE: Non-alcoholic fatty liver disease (NAFLD) is worldwide recognized as the most common cause of chronic liver disease. Current NAFLD clinical management relies on lifestyle change, nevertheless, the importance of the genetic make-up on liver damage and the possible interactions with diet are still poorly understood. The aim of the study was to evaluate the influence of the SH2B1 rs7359397 genetic variant on changes in body composition, metabolic status and liver health after 6-month energy-restricted treatment in overweight/obese subjects with NAFLD. In addition, gene-treatment interactions over the course of the intervention were examined. METHODS: The SH2B1 genetic variant was genotyped in 86 overweight/obese subjects with NAFLD from the FLiO study (Fatty Liver in Obesity study). Subjects were metabolically evaluated at baseline and at 6-months. Liver assessment included ultrasonography, Magnetic Resonance Imaging, elastography, a lipidomic test (OWL®-test) and specific blood liver biomarkers. Additionally, body composition, general biochemical markers and dietary intake were determined. RESULTS: Both genotypes significantly improved their body composition, general metabolic status and liver health after following an energy-restricted strategy. Liver imaging techniques showed a greater decrease in liver fat content (- 44.3%, p < 0.001) and in serum ferritin levels (p < 0.001) in the carriers of the T allele after the intervention. Moreover, lipidomic analysis, revealed a higher improvement in liver status when comparing risk vs. no-risk genotype (p = 0.006 vs. p = 0.926, respectively). Gene-treatment interactions showed an increase in fiber intake and omega-3 fatty acid in risk genotype (p interaction = 0.056 and p interaction = 0.053, respectively), while a significant increase in MedDiet score was observed in both genotype groups (p = 0.020). Moreover, no-risk genotype presented a relevant decrease in hepatic iron as well as in MUFA intake (p = 0.047 and p = 0.034, respectively). CONCLUSION: Subjects carrying the T allele of the rs7359397 polymorphism may benefit more in terms of hepatic health and liver status when prescribed an energy-restricted treatment, where a Mediterranean dietary pattern rich in fiber and other components such as omega-3 fatty acids might boost the benefits. TRIAL REGISTRATION: The Fatty Liver in Obesity was approved by the Research Ethics Committee of the University of Navarra and retrospectively registered (NCT03183193; www.clinicaltrials.gov ); June 2017.

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.

Modeling of an integrative prototype based on genetic, phenotypic, and environmental information for personalized prescription of energy-restricted diets in overweight/obese subjects.

BACKGROUND: Interindividual variability in weight loss and metabolic responses depends upon interactions between genetic, phenotypic, and environmental factors. OBJECTIVE: We aimed to model an integrative (nutri) prototype based on genetic, phenotypic, and environmental information for the personalized prescription of energy-restricted diets with different macronutrient distribution. METHODS: A 4-mo nutritional intervention was conducted in 305 overweight/obese volunteers involving 2 energy-restricted diets (30% restriction) with different macronutrient distribution: a moderately high-protein (MHP) diet (30% proteins, 30% lipids, and 40% carbohydrates) and a low-fat (LF) diet (22% lipids, 18% proteins, and 60% carbohydrates). A total of 201 subjects with good dietary adherence were genotyped for 95 single nucleotide polymorphisms (SNPs) related to energy homeostasis. Genotyping was performed by targeted next-generation sequencing. Two weighted genetic risk scores for the MHP (wGRS1) and LF (wGRS2) diets were computed using statistically relevant SNPs. Multiple linear regression models were performed to estimate percentage BMI decrease depending on the dietary macronutrient composition. RESULTS: After energy restriction, both the MHP and LF diets induced similar significant decreases in adiposity, body composition, and blood pressure, and improved the lipid profile. Furthermore, statistically relevant differences in anthropometric and biochemical markers depending on sex and age were found. BMI decrease in the MHP diet was best predicted at ∼28% (optimism-corrected adjusted R2 = 0.279) by wGRS1 and age, whereas wGRS2 and baseline energy intake explained ∼29% (optimism-corrected adjusted R2 = 0.287) of BMI decrease variability in the LF diet. The incorporation of these predictive models into a decision algorithm allowed the personalized prescription of the MHP and LF diets. CONCLUSIONS: Different genetic, phenotypic, and exogenous factors predict BMI decreases depending on the administration of a hypocaloric MHP diet or an LF diet. This holistic approach may help to personalize dietary advice for the management of excessive body weight using precision nutrition variables.This trial was registered at clinicaltrials.gov as NCT02737267.

The regulation of inflammation-related genes after palmitic acid and DHA treatments is not mediated by DNA methylation.

Fatty acids (FAs) are known to participate in body inflammatory responses. In particular, saturated FAs such as palmitic acid (PA) induce inflammatory signals in macrophages, whereas polyunsaturated FAs, including docosahexaenoic acid (DHA), have been related to anti-inflammatory effects. Several studies have suggested a role of fatty acids on DNA methylation, epigenetically regulating gene expression in inflammation processes. Therefore, this study investigated the effect of PA and DHA on the inflammation-related genes on human macrophages. In addition, a second aim was to study the epigenetic mechanism underlying the effect of FAs on the inflammatory response. For these purposes, human acute monocytic leukaemia cells (THP-1) were differentiated into macrophages with 12-O-tetradecanoylphorbol-13-acetate (TPA), followed by an incubation with PA or DHA. At the end of the experiment, mRNA expression, protein secretion, and CpG methylation of the following inflammatory genes were analysed: interleukin 1 beta (IL1B), tumour necrosis factor (TNF), plasminogen activator inhibitor-1 (SERPINE1) and interleukin 18 (IL18). The results showed that the treatment with PA increased IL-18 and TNF-α production. Contrariwise, the supplementation with DHA reduced IL-18, TNF-α and PAI-1 secretion by macrophages. However, the incubation with these fatty acids did not apparently modify the DNA methylation status of the investigated genes in the screened CpG sites. This research reveals that PA induces important pro-inflammatory markers in human macrophages, whereas DHA decreases the inflammatory response. Apparently, DNA methylation is not directly involved in the fatty acid-mediated regulation of the expression of these inflammation-related genes.

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.

Do the Effects of Resveratrol on Thermogenic and Oxidative Capacities in IBAT and Skeletal Muscle Depend on Feeding Conditions?

The aim of this study was to compare the effects of mild energy restriction and resveratrol on thermogenic and oxidative capacity in interscapular brown adipose tissue (IBAT) and in skeletal muscle. Rats were fed a high-fat high-sucrose diet for six weeks, and divided into four experimental groups fed a standard diet: a control group, a resveratrol-treated group, an energy-restricted group and an energy-restricted group treated with resveratrol. Weights of IBAT, gastrocnemius muscle and fat depots were measured. Activities of carnitine palmitoyltransferase (CPT) and citrate synthase (CS), protein levels of sirtuin (SIRT1 and 3), uncoupling proteins (UCP1 and 3), glucose transporter (GLUT4), mitochondrial transcription factor (TFAM), nuclear respiratory factor (NRF1), peroxisome proliferator-activated receptor (PPARα) and AMP activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator (PGC1α) activation were measured. No changes in IBAT and gastrocnemius weights were found. Energy-restriction, but not resveratrol, decreased the weights of adipose depots. In IBAT, resveratrol enhanced thermogenesis activating the SIRT1/PGC1α/PPARα axis. Resveratrol also induced fatty acid oxidation and glucose uptake. These effects were similar when resveratrol was combined with energy restriction. In the case of gastrocnemius muscle, the effects were not as clear as in the case of IBAT. In this tissue, resveratrol increased oxidative capacity. The combination of resveratrol and energy restriction seemingly did not improve the effects induced by the polyphenol alone.

Effects of perinatal diet and prenatal stress on the behavioural profile of aged male and female rats.

The present work studies whether chronic prenatal stress (PS) influences the long-term sex-dependent neuropsychological status of offspring and the effects of an early dietary intervention in the dam. In addition, dams were fed with either a high-fat sugar diet (HFSD) or methyl donor supplemented diet (MDSD). PS procedure did not affect body weight of the offspring. MDSD induced decreases in body weight both in male and female offspring (1 month) that were still present in aged rats. HFSD induced an increase in body weight both in male and female offspring that did not persist in aged rats. In the Porsolt forced swimming test, only young males showed increases in immobility time that were reversed by MDSD. In old female rats (20 months), PS-induced cognitive impairment in both the novel object recognition test (NORT) and in the Morris water maze that was reversed by MDSD, whereas in old males, cognitive impairments and reversion by MDSD was evident only in the Morris water maze. HFSD induced cognitive impairment in both control and PS old rats, but there was no additive effect of PS and HFSD. It is proposed here that the diversity of symptoms following PS could arise from programming effects in early brain development and that these effects could be modified by dietary intake of the dam.

Pterostilbene-induced changes in gut microbiota composition in relation to obesity.

SCOPE: Nutritional interventions based on the use of natural bioactive compounds might offer new possibilities for reshaping obesity-associated bacterial dysregulation or dysbiosis and improving health. We evaluated whether pterostilbene supplementation could induce changes in gut microbiota composition and whether these modifications were associated with improvements in metabolic variables. METHODS AND RESULTS: Zucker (fa/fa) rats were given a standard diet supplemented (n = 10) or not (n = 9) with pterostilbene (15 mg/kg body weight/day) by oral gavage for 6 weeks. Faucal samples at the beginning and at the end of the intervention period were analyzed by Illumina Mi-Seq sequencing approach. Pterostilbene exerted protective antiobesity effects, improved metabolic function (insulin sensitivity), and induced structural changes in gut microbiota composition. A decrease in the levels of Firmicutes and an increase in Verrucomicrobia phyla were detected in the pterostilbene-treated group. Bacterial species belonging to genera Akkermansia and Odoribacter were also increased. A strong inverse correlation between Akkermansia muciniphila and body weight was evidenced. Odoribacter splanchnicus showed a negative correlation with adiposity. CONCLUSION: Pterostilbene modifies intestinal bacteria composition toward a healthier microbial profile and suggests that the antiobesity effects induced in Zucker rats could be associated with an enrichment of the mucin-degrading bacterial members, namely Akkermansia and Odoribacter genus.

Methyl donor supplementation in rats reverses the deleterious effect of maternal separation on depression-like behaviour.

Adverse early life events are associated with altered stress responsiveness and metabolic disturbances in the adult life. Dietary methyl donor supplementation could be able to reverse the negative effects of maternal separation by affecting DNA methylation in the brain. In this study, maternal separation during lactation reduced body weight gain in the female adult offspring without affecting food intake, and altered total and HDL-cholesterol levels. Also, maternal separation induced a cognitive deficit as measured by NORT and an increase in the immobility time in the Porsolt forced swimming test, consistent with increased depression-like behaviour. An 18-week dietary supplementation with methyl donors (choline, betaine, folate and vitamin B12) from postnatal day 60 also reduced body weight without affecting food intake. Some of the deleterious effects induced by maternal separation, such as the abnormal levels of total and HDL-cholesterol, but especially the depression-like behaviour as measured by the Porsolt test, were reversed by methyl donor supplementation. Also, the administration of methyl donors increased total DNA methylation (measured by immunohistochemistry) and affected the expression of insulin receptor in the hippocampus of the adult offspring. However, no changes were observed in the DNA methylation status of insulin receptor and corticotropin-releasing hormone (CRH) promoter regions in the hypothalamus. In summary, methyl donor supplementation reversed some of the deleterious effects of an early life-induced model of depression in rats and altered the DNA methylation profile in the brain.

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.

Helichrysum and Grapefruit Extracts Boost Weight Loss in Overweight Rats Reducing Inflammation.

Obesity is characterized by an increased production of inflammatory markers. High levels of circulating free fatty acids and chronic inflammation lead to increased oxidative stress, contributing to the development of insulin resistance (IR). Recent studies have focused on the potential use of flavonoids for obesity management due to their antioxidant and anti-inflammatory properties. This study was designed to investigate the antioxidant and anti-inflammatory effects of helichrysum and grapefruit extracts in overweight insulin-resistant rats. Thirty-eight male Wistar rats were randomly distributed in two groups: control group (n=8) and high-fat sucrose (HFS) group (n=30). After 22 days of ad libitum water and food access, the rats fed HFS diet changed to standard diet and were reassigned into three groups (n=10 each group): nonsupplemented, helichrysum extract (2 g/kg bw), and grapefruit extract (1 g/kg bw) administered for 5 weeks. Rats supplemented with both extracts gained less body weight during the 5-week period of treatment, showed lower serum insulin levels and liver TBARS levels. Leptin/adiponectin ratio, as an indicator of IR, was lower in both extract-administered groups. These results were accompanied by a reduction in TNFα gene expression in epididymal adipose tissue and intestinal mucosa, and TLR2 expression in intestinal mucosa. Helichrysum and grapefruit extracts might be used as complement hypocaloric diets in weight loss treatment. Both extracts helped to reduce weight gain, hyperinsulinemia, and IR, improved inflammation markers, and decreased the HFS diet-induced oxidative stress in insulin-resistant rats.

Modulation of hyperglycemia and TNFα-mediated inflammation by helichrysum and grapefruit extracts in diabetic db/db mice.

Type-2 diabetes is associated with a chronic low-grade systemic inflammation accompanied by an increased production of adipokines/cytokines by obese adipose tissue. The search for new antidiabetic drugs with different mechanisms of action, such as insulin sensitizers, insulin secretagogues and α-glucosidase inhibitors, has directed the focus on the potential use of flavonoids in the management of type-2 diabetes. Thirty six diabetic male C57BL/6J db/db mice were fed a standard diet and randomly assigned into four experimental groups: non-treated control, (n = 8); acarbose (5 mg per kg bw, n = 8); helichrysum (1 g per kg bw, n = 10) and grapefruit (0.5 g per kg bw, n = 10) for 6 weeks. The mRNA expression in pancreas, liver and epididymal adipose tissue was determined by RT-PCR. DNA methylation was quantified in epididymal fat using pyrosequencing. Mice supplemented with helichrysum and grapefruit extracts showed a significant decrease in fasting glucose levels (p < 0.05). A possible mechanism of action could be the up-regulation of liver glucokinase (p < 0.05). The antihyperglycemic effect of both extracts was accompanied by decreased mRNA expression of some proinflammatory genes (monocyte chemotactic protein-1, tumor necrosis factor-α, cyclooxygenase-2, nuclear factor-kappaB) in the liver and epididymal adipose tissue. The CpG3 site of TNFα, located 5 bp downstream of the transcription start site, showed increased DNA methylation in the grapefruit group compared with the non-treated group (p < 0.01). In conclusion, helichrysum and grapefruit extracts improved hyperglycemia through the regulation of glucose metabolism in the liver and reduction of the expression of proinflammatory genes in the liver and visceral fat. The hypermethylation of TNFα in adipose tissue may contribute to reduce the inflammation associated with diabetes and obesity.

DNA methylation pattern in overweight women under an energy-restricted diet supplemented with fish oil.

Dietary factors modulate gene expression and are able to alter epigenetic signatures in peripheral blood mononuclear cells (PBMC). However, there are limited studies about the effects of omega-3 polyunsaturated fatty acids (n-3 PUFA) on the epigenetic mechanisms that regulate gene expression. This research investigates the effects of n-3-rich fish oil supplementation on DNA methylation profile of several genes whose expression has been reported to be downregulated by n-3 PUFA in PBMC: CD36, FFAR3, CD14, PDK4, and FADS1. Young overweight women were supplemented with fish oil or control in a randomized 8-week intervention trial following a balanced diet with 30% energy restriction. Fatty acid receptor CD36 decreased DNA methylation at CpG +477 due to energy restriction. Hypocaloric diet-induced weight loss also reduced the methylation percentages of CpG sites located in CD14, PDK4, and FADS1. The methylation patterns of these genes were only slightly affected by the fish oil supplementation, being the most relevant to the attenuation of the weight loss-induced decrease in CD36 methylation after adjusting by baseline body weight. These results suggest that the n-3 PUFA-induced changes in the expression of these genes in PBMC are not mediated by DNA methylation, although other epigenetic mechanisms cannot be discarded.

Maternal methyl donors supplementation during lactation prevents the hyperhomocysteinemia induced by a high-fat-sucrose intake by dams.

Maternal perinatal nutrition may program offspring metabolic features. Epigenetic regulation is one of the candidate mechanisms that may be affected by maternal dietary methyl donors intake as potential controllers of plasma homocysteine levels. Thirty-two Wistar pregnant rats were randomly assigned into four dietary groups during lactation: control, control supplemented with methyl donors, high-fat-sucrose and high-fat-sucrose supplemented with methyl donors. Physiological outcomes in the offspring were measured, including hepatic mRNA expression and global DNA methylation after weaning. The newborns whose mothers were fed the obesogenic diet were heavier longer and with a higher adiposity and intrahepatic fat content. Interestingly, increased levels of plasma homocysteine induced by the maternal high-fat-sucrose dietary intake were prevented in both sexes by maternal methyl donors supplementation. Total hepatic DNA methylation decreased in females due to maternal methyl donors administration, while Dnmt3a hepatic mRNA levels decreased accompanying the high-fat-sucrose consumption. Furthermore, a negative association between Dnmt3a liver mRNA levels and plasma homocysteine concentrations was found. Maternal high-fat-sucrose diet during lactation could program offspring obesity features, while methyl donors supplementation prevented the onset of high hyperhomocysteinemia. Maternal dietary intake also affected hepatic DNA methylation metabolism, which could be linked with the regulation of the methionine-homocysteine cycle.

Helichrysum and grapefruit extracts inhibit carbohydrate digestion and absorption, improving postprandial glucose levels and hyperinsulinemia in rats.

Several plant extracts rich in flavonoids have been reported to improve hyperglycemia by inhibiting digestive enzyme activities and SGLT1-mediated glucose uptake. In this study, helichrysum ( Helichrysum italicum ) and grapefruit ( Citrus × paradisi ) extracts inhibited in vitro enzyme activities. The helichrysum extract showed higher inhibitory activity of α-glucosidase (IC50 = 0.19 mg/mL) than α-amylase (IC50 = 0.83 mg/mL), whereas the grapefruit extract presented similar α-amylase and α-glucosidase inhibitory activities (IC50 = 0.42 mg/mL and IC50 = 0.41 mg/mL, respectively). Both extracts reduced maltose digestion in noneverted intestinal sacs (57% with helichrysum and 46% with grapefruit). Likewise, both extracts inhibited SGLT1-mediated methylglucoside uptake in Caco-2 cells in the presence of Na(+) (56% of inhibition with helichrysum and 54% with grapefruit). In vivo studies demonstrated that helichrysum decreased blood glucose levels after an oral maltose tolerance test (OMTT), and both extracts reduced postprandial glucose levels after the oral starch tolerance test (OSTT). Finally, both extracts improved hyperinsulinemia (31% with helichrysum and 50% with grapefruit) and HOMA index (47% with helichrysum and 54% with grapefruit) in a dietary model of insulin resistance in rats. In summary, helichrysum and grapefruit extracts improve postprandial glycemic control in rats, possibly by inhibiting α-glucosidase and α-amylase enzyme activities and decreasing SGLT1-mediated glucose uptake.

Transcriptomic and epigenetic changes in early liver steatosis associated to obesity: effect of dietary methyl donor supplementation.

Non-alcoholic fatty liver disease is a primary hepatic manifestation of obesity and an important adverse metabolic syndrome trait. Animal models of diet-induced obesity promote liver fat accumulation putatively associated with alterations in epigenetic profile. Dietary methyl donor-supplementation may protect against this disturbance during early developmental stages affecting the molecular basis of gene regulation. The aim of this study was to investigate the transcriptomic and epigenetic mechanisms implicated in liver fat accumulation as a result of an obesogenic diet and the putative preventive role of dietary methyl donors. Forty-eight male Wistar rats were assigned into four dietary groups for 8 weeks; control, control methyl-donor-supplemented with a dietary cocktail containing betaine, choline, vitamin B12 and folic acid, high-fat-sucrose and high-fat-sucrose methyl-donor-supplemented. Liver fat accumulation induced by a HFS diet was prevented by methyl donor supplementation in HFS-fed animals. A liver mRNA microarray, subsequently validated by real time-qPCR, showed modifications in some biologically relevant genes involved in obesity development and lipid metabolism (Lepr, Srebf2, Agpat3 and Esr1). Liver global DNA methylation was decreased by methyl donor supplementation in control-fed animals. Methylation levels of specific CpG sites from Srebf2, Agpat3 and Esr1 promoter regions showed changes due to the obesogenic diet and the supplementation with methyl donors. Interestingly, Srebf2 CpG23_24 methylation levels (-167 bp and -156 bp with respect to the transcriptional start site) correlated with HDLc plasma levels, whereas Esr1 CpG14 (-2623 bp) methylation levels were associated with body and liver weights and fat content. Furthermore HFS diet-induced liver fat accumulation was prevented by methyl donor supplementation. In conclusion, both obesogenic diet and methyl donor supplementation modified the mRNA hepatic profile as well as the methylation of specific gene promoters and total DNA.